Since women with climacteric syndrome have significantly lower serum levels of estradiol and other related hormones, hormone replacement therapies (HRT) such as estrogen are needed to lessen symptoms. However, HRT can often cause severe adverse effects that include many cancers and stroke. Therefore, new and novel approaches to relieve climacteric syndrome still need to be developed. The aim of this study was to identify biologically active peptides from calf spleen that are responsible for stimulating biosynthesis of steroid hormone and to explore the potential of isolated peptides as therapeutic agents for menopausal syndrome. The reverse phase HPLC system was used to isolate active compounds from calf spleen extract, a cell culture system was used to screen the activity of stimulating hormone secretion, and Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry was used for molecular weight determination. In the present study, two calf steroidogenic peptides, CSP-1 (MW; 4.655 kDa) and CSP-2 (MW; 8.331 kDa), were isolated and identified from calf spleen and may be putative climacteric syndrome therapeutic agents.