The cytotoxic and antifungal peptolides vioprolide A-D were discovered in 1996, however their mode-of-action has so far been unsolved. Anti-cancer effect of the most potent cytotoxic derivative vioprolide A (VioA) were evaluated, showing a prominent potency against human acute lymphoblastic leukemia (ALL) cells. In order to decipher the molecular mode-of-action, a VioA-derived photoprobe (VioA-P) was synthesized and utilized for MS-based AfBPP experiments. In addition thermal protein profiling (TPP) experiments with unmodified VioA were conducted for target discovery. TPP experiments revealed a strong thermal stabilization of two proteins after filtering. NOP14, the protein showing the strongest stabilization effect could further be verified as target with continuative biochemical experiments. As various protein-protein interactions with NOP14 are essential in ribosome biogenesis, e.g. with NOC4L and EMG1 for proper maturation of 18S rRNA and 40S assembly and export, the interactome of NOP14 was revealed by MS-based co-Immunoprecipitation. Our findings indicate, that VioA treatment leads to a loss of interaction between NOP14 and EMG1, whereas the NOP14-NOC4L interface remained unperturbed. These findings could be verified independently by western blot, also showing that overall protein expression of NOP14, EMG1 and NOC4L remained unaltered upon VioA treatment. Delocalization of EMG1 from the nucleus into the cytoplasm as consequence of missing interaction with NOP14 could be verified by immunostaining.