Manipulating autophagy is a promising strategy for treating cancer with several autophagy inhibitors shown to induce autophagic cell death. One of these, autophagonizer (APZ), induces apoptosis-independent cell death by binding an unknown target via an unknown mechanism. To identify APZ targets we developed a label-free drug affinity responsive target stability (DARTS) approach with a liquid chromatography/tandem mass spectrometry (LC-MS/MS) readout. Of 35 protein interactors, we identified APZBP as a key target protein of unmodified APZ in autophagy. Additionally, sequence coverage analysis in DARTS LC-MS proteome revealed that specific peptide fragments in the nucleotide binding domain (NBD, ATPase domain) of APZBP were protected by APZ in the pronase-treated proteome. APZBP inhibition through siRNA knockdown or by a specific inhibitor, inhibited autophagy in HeLa cells similar to APZ treatment, whereas APZBP overexpression overcame inhibition induced by APZ. Additionally, APZ exhibited significant synergism with TMZ (temozolomide), which has been used for chemotherapeutic agent of glioblastoma, in vitro and orthotopic mouse model. These findings demonstrate the potential of APZ to induce autophagic cell death and its development as a potent anticancer drug. Our study showed that combined DARTS and LC-MS/MS-based target identification of non-tagging bioactive small molecule of interest is effective for novel protein target identification and revealed a novel function for APZBP as cancer therapeutic target.