Pathologies including cancer, neurodegenerative, and cardiovascular diseases, are caused by the accumulation of misfolded/damaged proteins. Intracellular protein degradation mechanisms play a critical role in the clearance of these disease-causing proteins. Chaperone mediated autophagy (CMA) is a protein degradation pathway that employs chaperones to bind proteins, bearing a unique KFERQ-like motif, for delivery to a CMA-specific Lysosome Associated Membrane Protein 2a (LAMP2a) receptor for lysosomal degradation. To date, steady-state CMA function has been assessed by measuring LAMP2a protein expression. However, this does not provide information regarding CMA degradation activity. To fill this dearth of tools / assays to measure CMA activity in cells and tissues from preclinical models, we generated a CMA-specific fluorogenic substrate assay. Methods: A KFERQ-AMC [Lys-Phe-Asp-Arg-Gln-AMC(7-amino-4-methylcou-marin)] fluorogenic CMA substrate was synthesized from Solid-Phase Peptide Synthesis. KFERQ-AMC when cleaved via lysosomal hydrolysis causes AMC to release and fluoresce (Excitation:355 nm, Emission:460 nm). Using an inhibitor of lysosomal proteases, i.e., E64D [L-trans-Epoxy-succinyl-leucylamido(4-guanidino)butane)], responsible for cleaving CMA substrates, the actual CMA activity was determined. Essentially, CMA activity = (substrate)fluorescence - (substrate+E64D) fluorescence. To confirm specificity of the KFERQ sequence for CMA, scrambled peptides served as negative controls. Results: Heart, liver, and kidney lysates containing intact lysosomes were obtained from 4-month-old adult male mice (n=6 tissue samples/group). First, lysates incubated with KFERQ-AMC displayed a time dependent (0-5 hour) increase in AMC fluorescence vs. lysates incubated with scrambled peptides. These data validate the specificity of KFERQ for CMA. Of note, liver exhibited the highest CMA (6-fold; p kidney (2.4-fold) > heart (0.4-fold) at 5-hour. Second, E64D prevented KFERQ-AMC degradation, substantiating that KFERQ-AMC is degraded via lysosomes. Third, cleavage of KFERQ-AMC and resulting AMC fluorescence was inhibited in H9c2 cardiac cells transfected with LAMP2a vs. control siRNA. These data suggest LAMP2a is required for KFERQ degradation. Conclusion: We have generated a novel CMA activity assay for use in cells and tissues in a variety of experimental contexts.