Background: Seeds of Blepharis persica (Burm. f.) O. Kuntze are mentioned in Ayurveda to treat male reproductive functions debilities. Objectives: The present study aimed to evaluate the effect of extract and fraction prepared from seeds of B. persica, in vitro using TM3 Leydig cells and isolation of constituents from bioactive fraction. Materials and Methods: Methanol extract and ethyl acetate fraction prepared that were tested in vitro, using TM3 cell line for the testosterone production. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Ethyl acetate fraction of methanol extract was further taken up for detailed chemical investigations, considering biological potential. The fraction was subjected to column chromatography. The column was eluted with a step gradient of the proportion of methanol in chloroform for the separation of compounds. The isolated compounds were characterized using Fourier-transform infrared, nuclear magnetic resonance (1H and 13C), and mass spectroscopy for identification. Results: MTT assay performed using TM3 cells revealed that extract and fraction had a half-maximal inhibitory concentration value of more than 300 μg/ml. The methanol extract showed more than half-fold and ethyl acetate fraction of methanolic extract showed a one-fold increase in testosterone at a concentration of 150 μg/ml as compared to nontreated cells. Two constituents isolated from ethyl acetate fraction of methanolic extract were identified as apigenin 7-O-neohesperidoside and naringin using comparative spectral studies. Conclusion: The studies suggested that the plant extract and fraction could upregulate testosterone biosynthesis in Leydig cells. Two compounds isolated from the bioactive fraction might serve as a marker for assessment of the quality of plant material.