Nails can stably accumulate substances for long periods of time, thus providing retrospective information regarding drugs of abuse and pharmaceutical use. Nails have several advantages over the conventional matrices, such as blood and urine, including a longer detection window (months to years), non-invasive sample collection, and easy storage and transport. These aspects make nails a very interesting matrix for forensic and clinical toxicology. Because of the low concentrations of drugs of abuse and pharmaceuticals present in nails and the complexity of the keratinized matrix, analytical methods need to be more sensitive, and sample preparation is crucial. This review summarizes the literature regarding the detection and quantification of drugs of abuse and pharmaceuticals in nails, as well as the employed pre-analytical and analytical techniques. Additionally, the applications of nail analysis are reviewed. Finally, an overview of the challenges of nail analysis is provided, and guidelines for future research are proposed.