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Chemical Exchange Saturation Transfer (CEST) NMR spectroscopy is a powerful technique for characterizing these states; however, the complexity of data analysis and the computational cost of numerical fitting have hindered its widespread adoption. To address these challenges, we present ONEST (Optimized Novel Exchange Saturation Transfer), a user-friendly web server designed to automate and accelerate CEST analysis. ONEST utilizes a simultaneous multi-field fitting algorithm that leverages exact analytical solutions for two-state exchange (Baldwin model) rather than computationally intensive numerical integration. This approach incorporates a rigorous correction for radio-frequency (RF) field inhomogeneity and resolves parameter degeneracy by jointly fitting datasets acquired at distinct RF field strengths. Validation against synthetic datasets yielded reduced c2 values near unity (\u223c1.05), confirming that the analytical approach recovers kinetic parameters with accuracy comparable to full Bloch-McConnell simulations but at a fraction of the computational cost. Furthermore, application to the anti-HIV lectin OAA successfully characterized slow conformational exchange (kex = 279\u2009s\u22121) involving a minor population of 3%. By streamlining the extraction of kinetic and thermodynamic parameters, ONEST significantly lowers the technical barrier to entry, enabling a broader range of researchers to investigate protein dynamics at atomic resolution.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Contact<\/jats:title>\n                    <jats:p>dlee04@kbsi.re.kr<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Availability and implementation<\/jats:title>\n                    <jats:p>ONEST is available through the web server at http:\/\/onest.ai.kr.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/bioinformatics\/btag158","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T12:48:43Z","timestamp":1774529323000},"source":"Crossref","is-referenced-by-count":0,"title":["ONEST: a web-based platform for the rapid and robust analysis of protein excited states through CEST spectroscopy"],"prefix":"10.1093","volume":"42","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3800-0394","authenticated-orcid":false,"given":"Joonhyeok","family":"Choi","sequence":"first","affiliation":[{"name":"Center for Protein Structure and Drug Mechanism Research, Korea Basic Science Institute , Cheongju, 28119,","place":["Republic of Korea"]},{"name":"Department of Applied Pharmacy, College of Pharmacy, Chungbuk National University , Cheongju, 28160,","place":["Republic of Korea"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sang-Yeop","family":"Lee","sequence":"additional","affiliation":[{"name":"Center for Target-to-Therapeutics Research, Korea Basic Science Institute , Cheongju, 28119,","place":["Republic of Korea"]},{"name":"Department of Convergent Analytical Science, University of Science and Technology , Daejeon, 34113,","place":["Republic of Korea"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kyungdoe","family":"Han","sequence":"additional","affiliation":[{"name":"Department of Civil and Environmental Engineering, University of Wisconsin\u2014Madison , Madison, WI 53706,","place":["United States"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marta G","family":"Carneiro","sequence":"additional","affiliation":[{"name":"ZoBio B.V , Leiden, 2333 CH,","place":["The Netherlands"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kyoung-Seok","family":"Ryu","sequence":"additional","affiliation":[{"name":"Center for Protein Structure and Drug Mechanism Research, Korea Basic Science Institute , Cheongju, 28119,","place":["Republic of Korea"]},{"name":"Department of Convergent Analytical Science, University of Science and Technology , Daejeon, 34113,","place":["Republic of Korea"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3971-986X","authenticated-orcid":false,"given":"Donghan","family":"Lee","sequence":"additional","affiliation":[{"name":"Center for Protein Structure and Drug Mechanism Research, Korea Basic Science Institute , Cheongju, 28119,","place":["Republic of Korea"]},{"name":"Department of Convergent Analytical Science, University of Science and Technology , Daejeon, 34113,","place":["Republic of Korea"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2026,3,27]]},"reference":[{"key":"2026042409463841700_btag158-B1","doi-asserted-by":"publisher","first-page":"35","DOI":"10.1017\/S0033583514000122","article-title":"Visualizing transient dark states by NMR spectroscopy","volume":"48","author":"Anthis","year":"2015","journal-title":"Q Rev 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