Selective toxicity against prokaryotic over eukaryotic targets is the fundamental principle of safe antibacterial agents. In praxis, total selectivity is hard to achieve and, in fact, for many commonly used antibiotics side-effects on eukaryotic targets are known. For FQs, although not generally occurring, adverse events can be especially devastating and longlasting. Here, we applied (chemo-)proteomic approaches to decipher molecular mechanisms of FQ-derived human side-effects. Whole proteome analyses revealed significant protein dysregulation already after 3 h of treatment and pinpointed the reported mitochondrial toxicities of FQs to an arising proteotype with selective down-regulation of the complexes I and IV of the ETC in dependence of the FQ concentration. Subsequently, AfBPP and TPP were employed to identify protein off-targets.