Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.