Microsampling devices, such as dried blood spot (DBS) cards, offer significant advantages such as easy collection and low sample volume. However, their use poses challenges, including the effect of blood volume on analyses and the risk of contamination. The analytical validation of these methods requires a thorough evaluation of specific parameters, which can increase the number of required analyses compared to conventional methods. In the field of therapeutic drug monitoring, microsampling methods like DBS facilitate the collection of valuable information on drug interactions and patient metabolic capacities, thereby enhancing therapy efficacy and safety. Similarly, toxicological screening techniques benefit from the advantages of microsampling for faster and more comprehensive analysis. DBS analysis for doping detection offers advantages such as rapid and minimally invasive sample collection, as well as analyte stability at room temperature. However, it is subject to constraints related to sample preparation and volume. Recent advances in DBS technology have improved the detection and quantification of a wide range of drugs and psychoactive substances in clinical and forensic toxicology, offering more effective and stable methods despite some limitations related to sample collection and analysis. Finally, the application of DBS technology to environmental analysis enables the measurement of chemical concentration or presence in the human body, providing valuable information for assessing environmental exposures and contributing to the prevention of environmental health risks, particularly concerning trace elements, organic chemicals, and environmental exposure biomarkers such as tobacco smoke.