Quantification of pharmaceutical compounds in skin tissue is challenging because of low expected concentrations, small typical sample volumes, and the hard nature of the skin structure itself. This review provides a comprehensive overview of sample collection, sample homogenization and analyte extraction methods that have been used to quantify pharmaceutical compounds in skin tissue, obtained from animals and humans, using liquid chromatography-mass spectrometry. For each step in the process of sample collection to sample extraction, methods are compared to discuss challenges and provide practical guidance. Furthermore, liquid chromatographic-mass spectrometry considerations regarding the quality and complexity of skin tissue sample measurements are discussed, with emphasis on analyte recovery and matrix effects. Given that the true recovery of analytes from skin tissue is difficult to assess, the extent of homogenization plays a crucial role in the accuracy of quantification. Chemical or enzymatic solubilization of skin tissue samples would therefore be preferable as homogenization method.