Human aldo-keto reductases (AKRs) are mostly monomeric, cytosolic NADPH-dependent oxidoreductases involved in steroid metabolism, that catalyze reduction of a broad spectrum of carbonyl-containing substrates. Because AKRs are overexpressed in various cancers and are involved in resistance to chemotherapy via reduction and inactivation of anticancer drugs, design of selective AKR inhibitors is of great importance. The aim of this study was to express human AKR1C4 in E.coli, optimize a protein purification protocol and develop an enzyme activity assay using a known AKR1C substrate, 9,10-phenanthrenequinone (PQ), and inhibitor, ibuprofen. The pET28b(+)-AKR1C4 construct was provided from Dr. Chris Bunce. His-tagged AKR1C4 was purified using immobilized metal affinity and size-exclusion chromatography. As expected, the molecular weight of recombinant AKR1C4 estimated by SDS-PAGE was approximate 37 kDa and the purity of the obtained protein was sufficient for further studies. Kinetic fluorescence measurements of NADPH consumption confirmed that the enzyme was catalytically active. In the presence of AKR1C4, NADPH-dependent reduction of PQ resulted in a decrease in fluorescence over time, while no fluorescence change was observed in control reactions in the absence of enzyme. On the other hand, the slope of the curve in the presence of ibuprofen was lower vs. reaction control, suggesting enzyme inhibition by ibuprofen. Since imbalanced expression of human AKRs is associated with the development and progression of various diseases, such as hepatic cancer, this assay may be used for screening and identification of AKR1C4 inhibitors and design of potential therapeutics.