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Infection\u2010causing microorganisms often harbour multi\u2010drug resistance, requiring multiple tests for identification. Here, we present a multiplex functional assay using liquid atmospheric pressure (LAP) matrix\u2010assisted laser desorption\/ionization (MALDI) as next\u2010generation MALDI biotyping technology, which can accurately determine antibiotic resistance\/susceptibility within three hours from &lt;5\u00a0\u00b5L of bacterial culture, employing a beta\u2010lactam antibiotic panel. Strains with common resistance genes, including OXA\u201048, KPC\u20103, and VIM\u20101, as well as susceptible isolates, are easily and reliably classified. Concurrent with multi\u2010drug testing, the same bacterial sample provides species\u2010identifying lipid and protein profiles (up to 100% classification accuracy) and characterization through tandem mass spectrometry (MS\/MS) protein sequencing, facilitated by LAP\u2010MALDI's ability to generate multiply charged protein ions. Detection of multiple antibiotics, their degradation products and lipids\/proteins with high mass accuracy, along with the possibility of protein sequencing, offers new diagnostic possibilities for clinical microbiology and antimicrobial stewardship that are less probability\u2010based than conventional MALDI biotyping.<\/jats:p>","DOI":"10.1002\/advs.76927","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T18:46:35Z","timestamp":1785523595000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid Multiplex Antimicrobial Resistance Profiling and Bacterial Identification by LAP\u2010MALDI Mass Spectrometry Biotyping"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2652-9960","authenticated-orcid":false,"given":"Lily R.","family":"Adair","sequence":"first","affiliation":[{"name":"Department of Chemistry University of Reading  Reading Berkshire 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