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Raman spectroscopy has emerged as a powerful analytical tool for biochemical analysis owing to its ability to provide label-free, nondestructive, and chemically specific molecular information with minimal sample preparation. This review summarizes the principles and recent advances of Raman spectroscopy for the characterization of biomolecules and complex biological systems. We discuss its applications in the detection and mapping of major biochemical constituents, including proteins, lipids, nucleic acids, carbohydrates, and metabolites. Recent developments in Raman mapping have significantly enhanced the sensitivity and spatial resolution of biochemical investigations. The review further highlights the growing use of Raman-active probes and stable-isotope-based strategies and current challenges for monitoring metabolic processes and intracellular molecular dynamics. Collectively, this review provides an overview of the capabilities, applications, and emerging opportunities of Raman spectroscopy as a versatile platform for biochemical analysis.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00389","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:04:55Z","timestamp":1785852295000},"page":"2456-2469","source":"Crossref","is-referenced-by-count":1,"title":["Decoding the\nBiochemical Complexities Using Raman\nSpectroscopy"],"prefix":"10.1021","volume":"65","author":[{"given":"Jiro","family":"Karlo","sequence":"first","affiliation":[{"name":"Indian Institute of Technology Dharwad , , , ,","place":["Dharwad, Karnataka, India, 580011"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5688-1075","authenticated-orcid":true,"given":"Dhananjaya","family":"Giriraju","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Dharwad , , , ,","place":["Dharwad, Karnataka, India, 580011"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8901-2531","authenticated-orcid":true,"given":"Nilkamal","family":"Mahanta","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Dharwad , , , ,","place":["Dharwad, Karnataka, India, 580011"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9984-5385","authenticated-orcid":true,"given":"Surya Pratap","family":"Singh","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Dharwad , , , ,","place":["Dharwad, Karnataka, India, 580011"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,31]]},"reference":[{"key":"2026081808121817700_cit1","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1146\/annurev-anchem-091420-092323","article-title":"The Role of Raman Spectroscopy Within\nQuantitative Metabolomics","volume":"14","author":"Lima","year":"2021","journal-title":"Annu. 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