Background: Dioscorea deltoidea (Family: Dioscoreaceae) is a critically endangered medicinal plant widely used in traditional medicine and pharmaceutical industries for the preparation of appropriate drugs. The present study was conducted to investigate the effect of different elicitors such as salicylic acid (SA), methyl jasmonate (MeJa), jasmonic acid (JA) and abscisic acid (ABA) on the synthesis of diosgenin production in D. deltoidea shoot cultures. In addition, the effect of different precursors (squalene, β-sitosterol, and cholesterol) was also demonstrated. Results: Results showed that precursors significantly influenced diosgenin production as compared to elicitors. Application of SA (200 µM) showed highest diosgenin production (0.912% DW) following 4 h of incubation time, whereas MeJA (100 µM) exhibited 0.814% DW of diosgenin content following 8 h of incubation time. Among precursors, β-sitosterol at 200 µM produced maximum diosgenin content (1.006% DW) followed by 100 µM squalene (0.947% DW) harvested after 5th day of the precursor addition. Interestingly, cholesterol showed low diosgenin production, but significant than control cultures. Finally response surface methodology (RSM) was conducted to see the interactive effect of best elicitors and precursors concentration for better yield of diosgenin and biomass.Conclusions: The results revealed that exposure to different elicitors and precursors have a significant influence on accumulation of diosgenin in D. deltoidea shoot cultures. The optimized conditions for SA and β-sitosterol for the better yield of diosgenin and biomass were found to be 165 µM, 150 µM and 170 µM, 140 µM respectively.