Pancreatitis commonly occurs in humans, dogs, and cats. For both veterinary and human health-care professionals, measurement of serum pancreatic lipase concentration or activity provides useful support for a diagnosis of pancreatitis. In this Currents in One Health manuscript, we will discuss commonly used lipase assays in veterinary medicine, namely catalytic colorimetric and immunological lipase assays. We highlight potential diagnostic pitfalls associated with analytical specificity, assay validation, and sample condition interferences. Catalytic lipase assays may detect extrapancreatic lipases. In addition, we propose a decision tree for interpretation of lipase assays in the context of a clinical patient.