© 2021, The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature.Diosgenin is an important precursor of steroidal drugs and a number of biotechnology techniques, including gene transfer and elicitation studies, are used to produce it in higher volumes. With diosgenin levels of 0.2–0.9%, fenugreek is a useful source of this important substance. This study investigated the effect of yeast extract elicitation on diosgenin levels in suspension culture of fenugreek and determined the expression levels of important genes involved in the diosgenin pathway. Callus cultures from plants grown in vitro were used to establish cell suspension cultures. Yeast extract was applied at a concentration of 0.01 g/mL for 12 h and 24 h to elicit 13-day-old cultures, leading to 1.66- and 1.40-fold increase in diosgenin levels, respectively. Furthermore, we examined the expression of CYP86A2, an enzyme recently proposed to participate in C26 oxidation in sterol chain during diosgenin formation, and the enzyme genes of β-glucosidase and cycloartenol synthase, which are key enzymes in diosgenin formation. β-glucosidase and cycloartenol synthase gene expression decreased but CYP86A2 expression increased in cell suspension culture of fenugreek. With this study, we have evaluated the potential of yeast extract to increase diosgenin production in fenugreek cell suspension cultures. As a result, we have concluded that yeast extract is a promising potential elicitor for increasing diosgenin levels in fenugreek plants. Key Message: Yeast extract increased the amount of diosgenin content in fenugreek cell suspension cultures max 1.66 fold. 24-hour yeast extract treatment increased CYP86A2 gene expression and diosgenin content also.