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This study was aimed for assessment the effect of fenugreek extract and fenugreek NPs on the concentration of total protein in testicular tissues through application of two assays, Western blotting and Bicinchonic Acid. Totally, 60 Wister rats were selected, prepared and equally divided into three groups; negative control that remains without any treatment and given only distilled water daily; T1 that orally treated with a daily dose (100) mg/kg of extract fenugreek, and T2 that orally treated with a daily dose (100) mg/kg of fenugreek NPs. After an experimental period continued for 60 days, all study animals were euthanized with chloroform to collect of testis samples that subjected for homogenization and measurement of proteins concentration targeting of SDHA and CS proteins. The findings revealed that there is a significant increase in concentration of total protein in both fenugreek extract and fenugreek NP groups when compared with the value of control group. However, no significant variation was found between values of both fenugreek extract and fenugreek NP groups. Expression of mitochonderial expretion of SDHA protein was elevated in fenugreek extract group and fenugreek NP groups compared with control group. A comparison of values of treatment groups was revealed an elevation in values of fenugreek NP group more than fenugreek extract group. For CS protein, insignificant variation was reported between values of fenugreek extract and fenugreek NP groups when compared to value of control group. In conclusion, Western blotting demonstrated that is a powerful tool for detection and characterization of targeted proteins. Also, BCA revealed a high sensitivity and simple immunoassay for screening of increased total testicular protein. However, isolation of active components in fenugreek, and synthesizing of specific NPs based on one type of these components can provide new therapies with different characteristics. Also, the augmentation of fenugreek NPs and proper regulation of steroidogenesis and mitochondrial biogenesis related genes is notably need to be furthermore studied.