Homogeneous assays are characterized by rapidity, low cost, and simple workflows. However, relatively few specialized homogeneous platforms have garnered significant use in biological studies. Inconsistencies in matrix interferences, limited multiplexability, and the requirement for specialized instrumentation are among the various reasons for delayed acceptance. Recently, we have shown that DNA-driven protein assays using thermofluorimetric analysis (TFA) can limit matrix interference and promote multiplexing, all while requiring only a standard qPCR instrument for readout. Here, we show that homogeneous, one step (mix-and-read) TFA methods can be extended to the analysis of both a small molecule second messenger, cyclic adenosine monophosphate (cAMP), and a downstream cell-secreted hormone, insulin. Differential thermal analysis of DNA melting in these assays allowed analytical discrimination of background and signal without physical separation. The direct-readout, differential nature of TFA also promoted assay consistency and minimized calibration burden; analyte response curves were shown to be highly repeatable for up to 7 months. TFA protocols were validated by homogeneous quantification of both cAMP and insulin from single pancreatic islets undergoing a variety of treatments (glucose, KCl, glucose-responsive insulinotropic peptide (GIP), forskolin) that act upon glucose transporters, potassium and calcium channels, and G-protein-coupled receptors to modulate exocytosis. The results of this study suggest that TFA should be applicable to homogeneous quantification of a variety of small molecule messengers and protein analytes with standard instrumentation, thereby simplifying workflows in studies of cell-signaling cascades.