In this study, we examined the effect of plant growth regulators (2, 4-D, 6-BAP) and steroidal hormone (17-beta-estradiol) on a quantity of diosgenin in callus tissue of Polygonatum verticillatum (L.) All. The callus grown in in vitro conditions on MS medium with: 1/1, 2/2, 5/5, 10/10 ([mg/dm3] 2,4-D/[mg/dm3] 6-BAP) and 1/1/1, 2/2/2, 5/5/5, 10/10/10 ([mg/dm3] 2,4-D/[M/dm3] 17beta-estradiol/[mg/dm3] 6-BAP) was used for the densytometric analysis to determine the presence of diosgenin. The content of diosgenin ranged from 0,0642 to 1,1458%. An increase in diosgenin content due to addition of 6-BAP and 2,4-D was observed. The developed procedure was estimated by statistical analysis which indicates that it can be used for routine analysis.