Main conclusion Changes in proteome level as a result of methyl jasmonate and cholesterol treatment were investigated. The identified proteins were often involved in response to stress caused by various treatments. Furthermore, 18 proteins were expressed in treatmentspecific manner. In this study, the fenugreek plants were treated with methyl jasmonate (as an elicitor) and cholesterol (as a precursor of steroids and steroidal saponins) to check reaction at the level of the proteome to stress and to investigate steroidal saponin (diosgenin) biosynthesis. Proteins were separated by two-dimensional electrophoresis (2-DE) and identified by MALDI-ToF/ToF followed by database searches using Mascot search engine. Totally, 63 and 41 protein spots were differentially expressed after methyl jasmonate and cholesterol treatment, respectively. These proteins were classified into seven groups: photosynthesis, energy, metabolism, protein metabolism, secondary metabolism, stress and defense, and other. We found that 9 proteins were responsive to all treatments, and 18 proteins expressed in treatment-specific manner. Higher level of photosynthetic proteins sensitive to both biotic and abiotic stimuli was detected. In addition, proteins related to the stress (especially oxidative) and defense, protein, and secondary metabolism were overexpressed. The results indicate that methyl jasmonate and cholesterol elicited a defense reaction at the proteome level as a response to stress. The usefulness of 2-DE method for identification of proteins related with species-specific metabolic pathways is restricted. Integration of transcriptome data with proteomic analysis improved annotation process.