Abstract: A possibility of using an oil-in-water microemulsion of the composition 3% of sodium dodecyl sulfate, 8% of n-butanol, 1% of ethyl acetate, and 88% of water (by weight) as an extractant for the extraction and preconcentration of steroid hormones cortisol, cortisone, corticosterone, 11-deoxycortisol, and 11‑deoxycorticosterone from aqueous solutions and urine samples followed by their reversed-phase HPLC analysis with spectrophotometric detection is shown. In optimizing the extraction conditions, the nature of the oil, the time and temperature of extraction, and the time of the separation of the microemulsion were varied. Magnesium chloride was used to destroy the microemulsion. The concentration factors for steroid hormones increase with an increase in the hydrophobicity of the analytes. The limits of detection for steroids were 0.5–3 ng/mL, and concentration factors were 8–20.