Cytidine deaminase (CDA) and deoxycytidine monophosphate deaminase (DCTD) play crucial roles in pyrimidine metabolism, affecting DNA synthesis, cell cycle progression, and the efficacy of numerous nucleoside analog-based chemotherapeutics. Given their significance, accurate and sensitive measurement of their enzymatic activity is paramount for both fundamental biochemical research and clinical applications. This review provides a comprehensive overview of the methodologies used to assess CDA and DCTD activity, both established and emerging. We systematically categorize and discuss various approaches, including spectrophotometric, fluorimetric, liquid chromatography-based (Ultraviolet-Visible, fluorescence, and mass spectrometry), radiometric, and cell-based assays. For each method, we present its underlying principles, advantages, and limitations. Furthermore, we draw comparisons across the techniques to highlight their suitability for specific research questions.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry and Czech Advanced Technology and Research Institute Palacký University Olomouc Hněvotínská 5 779 00 Olomouc Czech Republic

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University Olomouc Hněvotínská 5 779 00 Olomouc Czech Republic

Laboratory of Inherited Metabolic Disorders Department of Clinical Chemistry Palacký University and University Hospital Olomouc Zdravotníků 248 7 779 00 Olomouc Czech Republic

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