Detection of changes in phosphorylation of cell signaling molecules using flow cytometry is termed "phosphoflow" or "phospho-flow cytometry". Phosphoflow has wide application for basic research into the mechanics of cell signaling, for evaluating aberrant signaling in cancerous cells and tissues, for studying efficacy or off-target effects during drug and vaccine development, and for functional assessment of pathogenic variants of genes that are known to play a role in development or function of the immune system. Phosphoflow has not been widely adopted in clinical laboratories owing to the challenges with developing and validating robust assays consistent with clinical laboratory regulatory standards. This review provides a brief overview of the utility of phosphoflow and points of consideration for development and validation of phosphoflow assays for diagnostic use, with a focus on inborn errors of immunity.