Chapter 26 In Vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells Elena Seranova, Carl Ward, Miruna Chipara, Tatiana R. Rosenstock, and Sovan Sarkar Abstract Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of aggregation-prone proteins and unwanted organelles. Dysregulation of autophagy underlying diverse human diseases reduces cell viability, whereas stimulation of autophagy is cytoprotective in a number of transgenic disease models including neurodegenerative disorders. Thus, therapeutic exploitation of autophagy is considered a potential treat- ment strategy in certain human diseases, and therefore, chemical inducers of autophagy have tremendous biomedical relevance. In this review, we describe the in vitro screening platforms to identify autophagy modulators in mammalian cells using various methodologies including fluorescence and high-content imaging, flow cytometry, fluorescence and luminescence detection by microplate reader, immunoblotting, and immunofluorescence. The commonly used autophagy reporters in these screening platforms are either based on autophagy marker like LC3 or autophagy substrate such as aggregation-prone proteins or p62/SQSTM1. The reporters and assays for monitoring autophagy are evolving over time to become more sensitive in measuring autophagic flux with the capability of high-throughput applications for drug discovery. Here we highlight these developments and also describe the stringent secondary autophagy assays for characterizing the autophagy modulators arising from the primary screen. Since autophagy is implicated in myriad human physiological and pathological conditions, these technologies will enable identifying novel chemical modulators or genetic regulators of autophagy that will be of biomedical and fundamental importance to human health. Key words Autophagy, Autophagy substrates, Autophagy modulators, Autophagy drug discovery, LC3, p62, Aggregation-prone protein, Chemical screen, Screening platform 1 Introduction Macroautophagy (herein referred to as autophagy) is the main disposal route for aggregation-prone protein and damaged orga- nelles in the cells. This evolutionarily conserved intracellular pro- teolytic pathway is essential for cellular homeostasis and organismal survival. Multiple lines of evidence have designated autophagy primarily as a cell survival mechanism [1]. Owing to its vital Nicholas Ktistakis and Oliver Florey (eds.), Autophagy: Methods and Protocols, Methods in Molecular Biology, vol. 1880, https://doi.org/10.1007/978-1-4939-8873-0_26, © Springer Science+Business Media, LLC, part of Springer Nature 2019 389