FQs inhibit the bacterial topoisomerase IV and gyrase, two enzymes essential for DNA replication. A crucial motif for the inhibition of both enzymes is the β-keto acid moiety of FQs, which acts as a chelator of active-site Mg 2+ ions. The β-keto acid of FQs binds via water-metal ion bridges to highly-conserved serine and acidic residues in the subunits A of the type-II topoisomerases. Consequently, these residues are most frequently mutated in FQ-resistant bacteria, defining the quinolone resistance-determining region (QRDR). Although a large variety of FQs were developed, only a few are used in human therapy against bacterial infections. Safety concerns led to the withdrawal of various members, such as Temafloxacin in 1992 and Grepafloxacin in 1999. Currently still marketed and commonly used derivatives, such as Ciprofloxacin (Cipro) and Levofloxacin (Levo), are usually welltolerated, however, severe, disabling and potentially long-lasting or permanent adverse effects are well documented. Potential side-effects include tendinopathy and tendon rupture, aortopathy, neuropathy , and various other adverse reactions, including CNS events. In 2015, the FDA termed this set of adverse effects the "Fluoroquinoloneassociated disability" (FQAD) and recommended to limit the use of FQs to infections insensitive to other alternative antibiotics. The reason for the increased sensitivity of some patients to FQs is not completely understood yet. A common theory is arising toxicity due to drug accumulation or concomitant prescriptions. Various mechanistic studies have demonstrated that FQs are strong metal chelators, impairing different enzymatic reactions due to cofactor complexation, such as the inhibition of irondependent dioxygenases, potentially leading to epigenetic effects. Likewise, integrin signaling, important for cell-cell and cell-extracellular matrix adhesion, and prolyl 4hydroxylases, involved in collagen maturation and modification, were reported to be affected via divalent ion coordination of FQs, suggesting multiple factors that could contribute to toxicity. Moreover, FQ treatment of human and animal tenocytes and human aortic myofibroblasts led to collagen degradation and increases in matrix metalloprotease (MMP) expression and activity. Both in tendons and ligaments, as well as in the aorta, collagen I and III are the major structural components. Mechanisms, such as mitochondrial dysfunction and increased oxidative stress are general consequences of FQ treatment. Also, various other theories of how FQs lead to adverse effects have been proposed, including, but not limited to, GABAA receptor inhibition, inhibition of mitochondrial topoisomerases, as well as α4β2 nicotinic acetylcholine receptor inhibition by Ciprofloxacin. Most reported studies focused on symptoms, indirect or downstream effects of FQ treatment and, to the best of our knowledge, no direct, unbiased and global off-target identification study to decipher the fundamental molecular origins of FQ-derived toxicity in human cells has been performed yet. To get insights into the molecular mechanisms of FQ-associated adverse effects, we here focused on the most commonly prescribed FQs Ciprofloxacin and Levofloxacin (Figure ) and applied (chemical-) proteomic methodologies to elucidate protein off-targets in human cells. For this we selected a two-tiered strategy based on thermal proteome profiling (TPP) as well as affinity-based protein profiling (AfBPP) with tailored FQ-derived photo-crosslinker probes. Among the identified targets, two crucial mitochondrial enzymes isocitrate dehydrogenase isoform 2 (IDH2) and apoptosis-inducing factor mitochondrial 1 (AIFM1), important for mitochondrial metabolism and respiratory chain biogenesis , respectively, were validated for FQ-dependent effects on their activity. Corresponding whole proteome analysis and biochemical assays of short and long-term FQ treatment confirmed major alterations in the respiratory chain complex I and IV as well as shifted NADPH levels corroborating the impaired cellular function of these target proteins. In light of rising antibiotic resistances and the dried-up antibiotic pipeline, especially for Gramnegative bacteria, this is of significant importance to maximize the feasibility of FQs for treatment of bacterial infections and facilitate the development of new and safer FQ generations.