Abstract Aim: To determine the inhibitory potential of 2 new fluoroquinolones, caderofloxacin and antofloxacin, together with 4 marketed fluoroquinolones, moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin, on the activity of cytochrome P450 isoforms 1A2 (CYP1A2) and 2C9 (CYP2C9). Methods: Probe substrates, phenacetin (CYP1A2), and tolbutamide (CYP2C9) were incubated with human liver microsomes and the metabolites were analyzed by liquid chromatography/mass spectrometry using electrospray ionization in positive or negative mode. Glipizide was used as the internal standard in both modes. The inhibitory potential of fluoroquinolones on CYP1A2 and CYP2C9 was investigated. Results: The IC50 values (mol/L) determined with the cocktail were in agreement with individual probe substrates (α-naphthoflavone: 0.27 vs 0.26; sulfaphenazole: 0.49 vs 0.37). Ciprofloxacin showed weak inhibition on both the activity of CYP1A2 (IC50 135 mol/L) and CYP2C9 (IC50 180 mol/L), whereas levofloxacin inhibited only CYP2C9 (IC50 210 mol/L). Caderofloxacin, antofloxacin, moxifloxacin, and gatifloxacin showed little or no inhibition on the activity of CYP1A2 or CYP2C9 when tested at comparable concentrations (0–200 mg/L). Conclusion: Caderofloxacin, antofloxacin, moxifloxacin, and gatifloxacin are negligible inhibitors to CYP1A2 and CYP2C9. The in vitro system can be used as a high-throughput model to screen similar compounds for the early identification of drug-drug interaction potential.