Fifty plants of Podophyllum hexandrum were collected from the interior ranges of western Himalaya of India from 8000 to 10000 ft above mean sea level for the analysis of podophyllotoxin content using HPLC. Two methods were used for the extraction of podophyllotoxin from powdered roots to standardize an extraction procedure for getting high yield of podophyllotoxin content. Extraction of podophyllotoxin was done efficiently in hot ethanol with continuous stirring. Highest podophyllotoxin content (7.84% on dry root wt basis) was obtained from P. hexandrum plants collected from Jalori Pass. In general, 24 per cent of the plant population from Jalori Pass contained higher amount of podophyllotoxin (4.0–7.84%). There was considerable reduction in the podophyllotoxin content in the roots of plants, planted at Palampur (4000 ft amsl) for one year. Embryogenic callus cultures obtained from root explants of P. hexandrum yielded 0.393% podophyllotoxin on dry wt basis.