Nucleosides and their corresponding mono-, di-, and triphosphates play important roles in maintaining cellular homeostasis. In addition, perturbations in this homeostasis can result in dysfunctional cellular processes that cause pathological conditions such as cancer and autoimmune diseases. This review article discusses contemporary research areas applying nucleoside analogs to probe the mechanistic details underlying the complexities of nucleoside metabolism at the molecular and cellular levels. The first area describes classic and contemporary approaches used to quantify the activity of nucleoside transporters, an important class of membrane proteins that mediate the influx and efflux of nucleosides and nucleobases. A focal point of this section is describing how biophotonic nucleosides are replacing conventional assays employing radiolabeled substrates to study the mechanism of these proteins. The second section describes approaches to understand the utilization of nucleoside triphosphates by cellular DNA polymerases during DNA synthesis. Emphasis here is placed on describing how novel nucleoside analogs such as 5-ethynyl-2'-deoxyuridine are being used to quantify DNA synthesis during normal replication as well as during the replication of damaged DNA. In both sections, seminal research articles relevant to these areas are described to highlight how these novel probes are improving our understanding of these biological processes. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. (C) 2015 Elsevier B.V. All rights reserved.