Dutasteride is a specific and selective inhibitor of both 5α-reductase isoforms used mainly in benign prostatic hyperplasia and lower urinary tract symptoms. Although the drug is extensively metabolized in humans, data on the concentrations of its main metabolites are lacking. There is also a lack of data on dutasteride stability in frozen plasma samples. Our method was used to determine dutasteride and its active metabolites: 4'-hydroxydutasteride, 6β-hydroxydutasteride, and 1,2-dihydrodutasteride in plasma after a single administration of 0.5 mg of dutasteride. We also assessed the long-term stability (two years in the freezer) of dutasteride in clinical samples. The developed method covered the range of 0.1-3.5 ng/mL for dutasteride and 0.08-1.2 ng/mL for 1,2-dihydrodutasteride, 4'-hydroxydutasteride, 6β-hydroxydutasteride. It was proved to be reliable as it met all validation criteria required by the European Medicine Agency for bioanalytical methods. 4'-hydroxydutasteride and 1,2-dihydrodutasteride concentrations in plasma were higher than 6β-hydroxydutasteride. Dutasteride was stable in the freezer for up to 2 years in clinical samples. Thus within 1014 days of storage (below - 65 °C), samples can be reanalyzed without the risk of unreliable results.