Nucleoside/tide analogs have played a key role in the treatment of viral infections caused by DNA viruses such as herpes virus and hepatitis B virus, as well as RNA viruses such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, and most recently the coronavirus SARS-CoV-2, the pathogen causing the COVID-19 pandemic. Studies over the past several decades have significantly expanded our knowledge on the potential off-target effects for this class of compounds, including inhibition of host nucleic acid polymerases such as human mitochondrial DNA polymerase γ (POL γ) and mitochondrial RNA polymerase (POLRMT) as well as perturbation of nucleotide metabolism, mitochondrial respiration, and dNTP/NTP pools. Importantly, suitable methods to evaluate the potential for these types of toxicities are currently available. This presentation will provide a historical review of the discovery of safe and efficacious antiviral nucleoside/tide analogs and lessons learned from failed clinical studies due to safety concerns. We will also discuss the utility of a panel of biochemical and cell-based assays to assess toxicity-liability in the early drug discovery stage.