A simple, sensitive and specific thin layer chromatography densitometric method has been developed for the simultaneous quantification of vincristine and vinblastine in the leaves of catharanthus roseus. The method involved simultaneous estimation of vincristine and vinblastine after resolving it by High Performance TLC on silica gel plate with toluene-methanol-diethylamine (8.75: 0.75: 0.5 v/v/v) as the mobile phase. The method was validated as per the ICH guidelines for precision (inter-day, intra-day, inter-system), robustness, accuracy, LOD and LOQ. The relationship between the concentration of standard solutions and the peak response was linear within the concentration range of 100ng/spot to 4000ng/spot for vincristine and 200ng/spot to 4000ng/spot for vinblastine. The method precision was found to be 0.77-1.78 (%RSD) and 1.24-2.13 (% RSD) for vincristine and vinblastine, respectively. Accuracy of the method was checked by recovery study conducted at three different levels and the average percentage recovery was found to be 100.21 % for vincristine and 99.99 % for vinblastine, respectively. The HPTLC method for the simultaneous quantification of vincristine and vinblastine was found to be simple, precise, specific, sensitive and accurate and can be used for routine analysis and quality control of raw material of C. roseus. and several unani and ayurvedic formulations containing as an ingredient.