Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.