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To address the question of whether established or experimental anticancer chemotherapeutics can exert their cytotoxic effects by autophagy, we performed a high-content screen on a set of cytotoxic agents. We simultaneously determined parameters of autophagy, apoptosis and necrosis on cells exposed to∼ 1400 compounds. Many agents induced a ‘pure’autophagic, apoptotic or necrotic phenotype, whereas less than 100 simultaneously induced autophagy, apoptosis and necrosis. A systematic analysis of the autophagic flux induced by the most potent 80 inducers of GFP-LC3 puncta among the NCI panel agents showed that 59 among them truly induced autophagy. The remaining 21 compounds were potent inducers of apoptosis or necrosis, yet failed to stimulate an autophagic flux, which were characterized as microtubule inhibitors. Knockdown of ATG7 was efficient in preventing GFP-LC3 puncta, yet failed …