$5{alpha}$-Dihydrotestosterone (DHT) is the primary active metabolite of testosterone, catalyzed by $5{alpha}$-reductase ($5{alpha}R$) in the skin, prostate, and liver. In this study, the $5{alpha}R$ activity in rat liver S9 fraction in the presence of a NADPH-generating system was evaluated and compared by gas chromatography-mass spectrometry (GC-MS)-based in vitro assays. Testosterone and a $5{alpha}R$ inhibitor, finasteride, were added to the S9 fractions and incubated at $37^{circ}C$ for 1 h. Both testosterone and DHT were quantitatively measured and compared with two different GC-MS-based steroid profiling techniques. DHT was not detected by conventional GC-MS analysis in the absence of finasteride when the concentration of testosterone in the S9 fraction was less than $0.2{mu}M$, whereas the isotope-dilution GC-MS (GC-IDMS) system was able to evaluate the $5{alpha}R$ activity. Because the S9 fraction contains more reactive enzymes and is easier to collect from tissues compared with a microsomal solution, the combination of the S9 fraction and GC-IDMS technique may be a promising assay for evaluating the $5{alpha}R$ activity in large-scale clinical studies.