The validated micellar electrokinetic chromatography (MEKC) was proposed for the determination of five steroid hormones in human urine samples. That technique allowed for the separation and quantification of cortisol, cortisone, corticosterone, testosterone, and epitestosterone and was sensitive enough to detect low concentrations of these searched steroids in urine samples at the range of 2-300 ng/mL. The proposed MEKC technique with solid-phase extraction (SPE) procedure was simple, rapid, and has been successfully applied as a routine procedure to analyze steroids in human urine samples. The MEKC method offered a potential in clinical routine practice because of the short analysis time (8 min), low costs, and simultaneous analysis of five endogenous hormones. Due to its simplicity, speed, accuracy, and high recovery, the proposed method could offer a tool to determine steroid hormones as potential biomarkers in biomedical investigations, what was additionally revealed with healthy volunteers.