Abstract <p>Modified nucleoside drugs are used in clinical practice to treat oncohematological diseases (nelarabine, fludarabine, cladribine, clofarabine) and viral infections (vidarabine, ribavirin, etc.) in the human body. The development of efficient technologies for the production of pharmaceutical substances is an important task for medical biotechnology. There are two main approaches to the synthesis of modified nucleosides: chemical and enzymatic. Traditional chemical synthesis technologies are often complex, labor-intensive, and environmentally unfriendly. As an alternative, enzymatic synthesis using nucleoside phosphorylases and other nucleic acid metabolism enzymes allows the synthesis of nucleosides in one pot with exceptional stereo- and regioselectivity. The desired compounds can be obtained either through enzymatic transglycosylation or multienzymatic cascades, which allow synthesis to begin from pentofuranoses or other readily available substrates. In some cases, the narrow substrate specificity of natural biocatalysts and low stability under industrial conditions can become an obstacle for the enzymatic synthesis of modified nucleosides. This paper offers an overview of the reported examples of the application of rational design and directed evolution approaches for the optimization of nucleoside production. The review focuses on both monoenzymatic variants (nucleoside phosphorylases) and multienzyme cascades for the synthesis of modified nucleosides.</p>