Enzymes are the catalysts for virtually every physiological process and thus provide a key to unlocking aberrant metabolism across a wide range of pathologies; including cancers. High-resolution maps of enzyme activity within tissues would therefore represent powerful tools for elucidating enzymatic functions in health and disease, and have potentially novel diagnostic applications. To-date however, no single imaging modality is able to routinely and simultaneously map the distribution and efficacy of multiple enzymes in situ. Mass spectrometry imaging has contributed to the understanding of many biological systems on the basis of showing where the molecules exist, however, the dynamic range is affected by the chemical complexity of the samples in the tissue sections being analysed. Here, we present a new mass spectrometry imaging (MSI)-based method for assaying the spatial distribution of enzymatic activity directly from tissue sections - potentially enabling the visualisation of specific enzymes by their activity rather than their molecular weight, or proteotypic peptide fragment(s). By depositing enzyme substrates on tissue sections that are subsequently analysed by MSI, high-resolution maps of enzyme activity and substrate specificity can be generated. Using phospholipases as model enzyme targets, we demonstrate that functional mass spectrometry imaging (fMSI) represents a new and generalizable method for visualising biological activity across tissues, which is in-principle applicable to any enzyme class with soluble, low molecular weight products.