Arbutin and quercetin are anti-cancer agents, extracted from leaves and flowers of Origanum majorana L which can be simultaneously estimated using a rapid, specific and sensitive high – performance thin layer chromatographic technique using silica gel G60F254 as the stationary phase and butyl acetate: methanol: formic acid: toluene (8: 1.5: 5: 0.5) as mobile phase for the separation. The method was validated for linearity, precision and recovery. Linearity was established in the range 300-900 ng/spot for arbutin and 150-450 ng/spot for quercetin, respectively. The % RSD values of repeatability of application, repeatability of measurement, intra-day and inter day precision studies of arbutin and quercetin were found to be below 1 for 500 and 250 ng/spot of arbutin and quercetin, respectively. The recovery of the drugs were found to be 99.8 and 100.2%, proves that the developed method was highly accurate. Hence the proposed validated method could be applied for the simultaneous analysis of arbutin and quercetin in methanolic extract of Origanum majorana L as well as for its marketed formulation.