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Liquid extraction surface analysis mass spectrometry (LESA‐MS) is a novel surface profiling technique that combines micro‐liquid extraction from a solid surface with nano‐electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA‐MS can provide drug‐ and metabolite‐specific distribution information. Here, we evaluate LESA‐MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI‐MSI. The spatial resolution of LESA‐MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also …