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BackgroundThe scientific validity of contemporary and alternative medicinal practices, such as Ayurveda and Siddha, holds significant importance in today’s context. Aim and ObjectiveThe present study employs gas chromatography-mass spectrometry (GC-MS) analysis to investigate a pain-relieving Ayurvedic oil, Dhanwantharam Thailam, aiming to establish correlations between its medicinal activity and the biomolecules it contains. Materials and MethodsProcured sample from a reputable Ayurvedic vendor in Chennai, Dhanwantharam Thailam underwent GC-MS analysis using standard procedures. Results and DiscussionThe resulting profile revealed the presence of crucial molecules like oleic acid, dodecanoic acid, 1,2,3-propanetriyl ester, ethenyl ester, and 9,12-octadecadienoyl chloride (Z, Z), aligning with the medicinal properties attributed to Dhanwantharam Thailam. ConclusionThe identification of these biomolecules supports the role of Dhanwantharam Thailamas an effective pain-relieving oil.