The evaluation of the relationships between the hormones involved in the urogenital tract cancer, including bladder, kidney, prostate, and testis, could prove important from diagnostic point of view. The determination of the steroid hormone profiles may likely provide a biomarker for discrimination of hormone-related diseases, as well as for differentiation of healthy volunteers from patients with cancer. The aim of the study was to demonstrate the changes in the steroid hormone profile (comprising corticosteroids, androgens and progesterone) in the urine of patients with the urogenital tract cancer versus urine from healthy subjects. A reliable analytical method based on liquid chromatography coupled with mass spectrometry was successfully applied to determine the urinary profiles of 6 endogenous steroids: cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone for 92 urogenital tract cancer patients and 100 healthy controls. The obtained data was further evaluated by in-depth chemometric analysis, including the applied standardized Kennard-Stone's algorithm to pre-process the data. Mann-Whitney U test revealed statistically significant (p <0.05) differences in concentration of androgens and progesterone in the case of bladder cancer for male and female population, for male also cortisol and cortisone levels were significantly increased. PCA analysis proved a reasonable trend for differentiating healthy and cancer patients, and finally, applying PLS-DA model we were able to correctly classify 80.56%of cancer patients. Our results indicate that steroid hormone profile determination could be a promising approach for early diagnosis of urogenital tract cancer. However our preliminary results require an extension both in patient number and steroid profile.