A simple, sensitive, specific, rapid and accurate high performance thin layer chromatographic (HPTLC) method has been developed for the quantification of podophyllotoxin and etoposide. Podophyllotoxin was quantified in the roots of Podophyllum hexandrum whereas etoposide in a marketed formulation. The method involved densitometric evaluation of both podophyllotoxin and etoposide after resolving it on silica gel plate using dichloromethane–methanol–formic acid (9.5:0.5:0.5 v/v/v) as the mobile phase. The method was validated for precision (inter-day, intra-day and inter-system), robustness, accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response (area) was linear within the concentration range of 150–2400 ng spot−1 for podophyllotoxin and 200–2000 ng spot−1 for etoposide. Instrumental precision was found to be 1.03–1.80 (% RSD) and 0.79–1.99 (% RSD) for podophyllotoxin and etoposide, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.71% for podophyllotoxin and 100.43% for etoposide, respectively. The HPTLC method for the quantification of podophyllotoxin and etoposide was found to be simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of P. hexandrum and several formulations containing these markers.