Unani system is a science which deals with the preventive and promotive aspects of health of human beings and health problems occurred by the Ecological and Environmental factors, which may vitiate humours i.e. Blood, Phlegm, Yellow bile and Black bile, the fluids circulating in the body vessels. There is considerable demand in drug testing for a specific and precise analytical method for the identification of alliin in various Unani formulations. A combination of high performance liquid chromatography and mass spectrometry (LC-MS/MS) will provide unambiguous fingerprint information for estimation of alliin in Unani formulations. The objective of the present investigation is to develop a simple, economical and reliable high performance liquid chromatography method for the quantification of alliin in Unani formulations. The validated method allows quantification of alliin in 1 – 100.00 μg/mL. The correlation coefficient was ≥0.9990 for the Alliin. The simplicity of the assay and rapid liquid-liquid extraction make it an attractive procedure in estimation of alliin in Unani formulations. The separation was achieved in C18 column and positive ion mode was used for detection in ESIMS detection.