Although the steroidogenesis in testis occur in a paracrine rather than in an endocrine, the comparative metabolic pathways between fetal and adult mice are not fully understood. Here, gas chromatography-mass spectrometry-based assay for profiling of 23 androgens, 7 estrogens, 13 corticoids, and 14 progestagens has been developed and applied to quantify their testis levels from both fetal and adult mice. Testis samples were purified with Oasis HLB solid-phase extraction and separated through a MXT-1 (30 m×0.25 mm I.D., 0.25 μm film thickness, Siltek-treated stainless steel) column prior to GC-triple quadrupole/MS (GCMS/MS) analysis. The devised assay showed a good linearity (r2> 0.991) with precisions (%CV) and accuracies (%bias) ranged from 1.1% to 15.4% and from 82% to 117%, respectively. All steroids detected were higher in adult testes, while Δ5-androstenediol (A-diol) was only quantitative in fetal testes, which mean the conversion of DHEA into A-diol may be dominantly occurred during fetal development. In addition, the metabolic ratios of 7α-hydroxylation of androstenedione and testosterone were significantly increased in fetal testes. The metabolic signatures of steroids in testes reflect the comparative steroidogenesis during developmental stages and may useful for monitoring the metabolic response to physiological conditions.