Autophagy is a major intracellular catabolic pathway induced in response to amino acid starvation. Recent findings implicate it in diverse physiological/pathophysiological events, such as protein and organelle turnover, development, aging, pathogen infection, cell death, and neurodegeneration. However, experimental methods to monitor this process in mammalian cells are limited because of the deficiency of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homolog of the yeast ubiquitin-like (UBL) protein Atg8, has been shown to selectively incorporate into the autophagosomal membrane, thus serving as a unique bona fide marker of autophagosomes in mammals. Thus, the autophagic activity can be largely determined by GFP-LC3/LC3, predominantly associated with autophagosomes (when LC3 is conjugated to phosphatidylethanolamine), both biochemically and microscopically. However, current methods to quantify autophagic activity using LC3 are time consuming, labor intensive, and require expertise in accurate interpretation. In this chapter we describe the use of flow cytometry and fluorescence-activated cell sorting (FACS) as a new assay designed to quantify autophagy in cells stably expressing GFP-LC3. Flow cytometry is a well-established technique for performing quantitative fluorescence measurements, allowing quick, accurate, and simultaneous determination of many parameters in cell subpopulations. Here flow cytometry and FACS were used to quantify the turnover of GFP-LC3 (reflecting an autophagic flux) as a reliable and simple assay to measure autophagic activity in living mammalian cells.