Human type 3 3Ξ±-hydroxysteroid dehydrogenase (3Ξ±HSD-3) is a member of the AKR1C subfamily of enzymes. Members of the AKR1C subfamily catalyze thereduction of aldehyde and keto groups of various endogenous and exogenoussubstrates including steroids, prostaglandins, and xenobiotics. AKR1Cenzymes work in an NADPH-dependent manner. A distinctive characteristic of3Ξ±HSD-3 is that it binds bile acids, which in return act as inhibitors of itsenzymatic activity. 3Ξ±HSD-3 is involved in the development of certainpathological conditions including several types of cancer, where itsoverexpression has been proven to lead to chemotherapy resistance. In thisthesis, a library of bile acid derivatives was put through in vitro screening forchanges in 3Ξ±HSD-3 enzymatic activity. For compounds that have showninhibitory potential towards 3Ξ±HSD-3, inhibition strength and specificity wereestimated utilizing IC50 measurement and 17Ξ²HSD-5 inhibition screening.Finally, in silico and crystallographic data were combined to propose astructural model of binding and inhibition of enzymatic activity. Severalcompounds were found to bind to the 3Ξ±HSD-3 active site, and this binding wasfavored by polar contacts between hydrophilic groups of tested compounds andamino acid residues in the active site of the enzyme. Small changes in the position of certain residues were noticed, and these changes improved the fitting of ligands to the active site of 3Ξ±HSD-3. This β€˜induced fitting’ of ligand to the active site of 3Ξ±HSD-3 has been observed by several research groups in the past. Results shown in this dissertation can lead to the synthesis of better inhibitors of 3Ξ±HSD-3, which could be used in fundamental science, but might as well lead to the development of new drugs that would serve as adjuvant therapy for the treatment of metastatic diseases and other conditions caused by 3Ξ±HSD-3 overexpression.