Autophagy has gained considerable attention in the last few years due to its importance in disease processes such as tumorigenesis and neurodegeneration. Significant efforts are ongoing to identify modulators of the autophagy pathway. Sequestosome (p62) and Microtubule-Associated Protein 1 Light Chain 3 (LC3) are key players in the autophagy process. LC3 is involved in formation of the autophagosomal membrane and p62 sequesters polyubiquitinated proteins in autophagosomes. To date, LC3 has been a frequently used marker for autophagy. The modification of LC3 by attachment of phosphatidylethanolamine is detectable by Western blotting, and its cellular localization at the autophagosomal membrane can be visualized using immunofluorescence microscopy. However, there is a lack of high-throughput methods for monitoring autophagy. For this reason, AlphaLISA® no-wash assays have been developed for the detection of endogenous levels of p62 and LC3B in cell lysates. The cellular model system used was HeLa cells treated with chloroquine to inhibit the final autophagy step - autolysosomal degradation. This inhibition leads to an accumulation of the autophagosome marker proteins. Both Western blotting and immunofluorescence were performed to validate the model system. In a typical experiment, the cells were treated overnight with 50 µM chloroquine, following which the AlphaLISA assay was initiated by direct addition of lysis buffer to the wells of the microplate. The analyte was simultaneously captured by a biotinylated antibody and antibody-conjugated Alpha acceptor beads. The assay was then completed by addition of Streptavidin-coupled Alpha Donor beads and read on an EnVision® Multimode Plate Reader. The levels of LC3B and p62 both increased upon treatment with chloroquine by 2 to 5-fold. The autophagy markers could be detected using as little as 1000 cells in a 384-well plate. These assays can accelerate the identification of therapeutics that modulate the autophagy process.