Abstract: | Enzymes are central components of most physiological processes, and are consequently implicated in various pathologies. High‐resolution maps of enzyme activity within tissues therefore represent powerful tools for elucidating enzymatic functions in health and disease. Here, we present a novel mass spectrometry imaging (MSI) method for assaying the spatial distribution of enzymatic activity directly from tissue. MSI analysis of tissue sections exposed to phospholipid substrates produced high‐resolution maps of phospholipase activity and specificity, which could subsequently be compared to histological images of the same section. Functional MSI thus represents a new and generalisable method for imaging biological activity in situ. |