Our understanding of autophagy has expanded tremendously in recent years, largely due to the identification of the many genes involved in the process, and the use of GFP-LC3 fusion proteins to visually monitor autophagosomes and autophagic activity both biochemically and microscopically [1, 2]. Recently, a novel fluorescent probe, Cyto-ID® Green autophagy dye, has been developed to facilitate the investigation of the autophagic process [3-5]. In this study, a novel method was performed using the Cellometer image cytometry in combination with Cyto-ID Green autophagy dye for detecting autophagy in live cells. First, Cyto-ID Green autophagy dye was validated by observing co-localization of the dye and RFP-LC3 in HeLa cells using fluorescence microscopy. Next, image-based and flow cytometry-based methods are benchmarked for measuring macroautophagic signals in nutrient-starved Jurkat cells. Autophagic signals of starved Jurkat cells induced with an autophagy inhibitor were also quantified and compared using the two instrument platforms . In order to establish the feasibility of employing the imaging-based workflow for drug discovery applications, a timecourse study of the induction of autophagy in Jurkat (suspension) and PC-3 (adherent) cells treated with rapamycin was undertaken [7, 8], demonstrating the ability to detect autophagy with a similar sensitivity as the starvation model. Finally, direct dose-response comparisons of two small molecule autophagy inducers, rapamycin and tamoxifen, were performed .