Biological tissue consists of populations of cells exhibiting different responses to pharmacological stimuli. To probe the heterogeneity of cell function, we propose a multiplexed approach based on real‐time imaging of the secondary messenger levels within each cell of the tissue, followed by extraction of the changes of single‐cell fluorescence over time. By utilizing a piecewise baseline correction, we were able to quantify the effects of multiple pharmacological stimuli added and removed sequentially to pancreatic islets of Langerhans, thereby performing a deep functional profiling for each cell within the islet. Cluster analysis based on the functional profile demonstrated dose‐dependent changes in statistical inter‐relationships between islet cell populations. We therefore believe that the functional cytometric approach can be used for routine quantitative profiling of the tissue for drug screening or pathological testing.