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Background: Autofluorescence (AF)-Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min., Objectives: To determine the accuracy of the AF-Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard., Methods: Skin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022-July 2023) were investigated with the AF-Raman instrument. The layers were measured when fresh, immediately after excision. The AF-Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622)., Results: AF-Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF-Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49-82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63-82). Only one of 12 false-negative cases was caused by misclassification by the AF-Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70-95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81-95)., Conclusions: The AF-Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens., Competing Interests: Conflicts of interest S.V., A.K., H.C.W. and I.N. hold a patent related to Raman microspectroscopy technology. R.A.B., A.S., S.O., S.E., K.E., S.G., A.P., K.D.W. and S.K. declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Association of Dermatologists.)