Cytochromes P450 (CYP) are subject to important interindividual variability in their activity due to genetic and environmental factors and some diseases. Limited human data support the idea that inflammation down-regulates CYP activities. Our study aimed to evaluate the impact of orthopedic surgery (acute inflammation model) on the activity of six human CYP. This prospective observational study was conducted in thirty patients who underwent elective hip surgery at the Geneva University Hospitals in Switzerland. The Geneva phenotyping cocktail containing caffeine, bupropion, flurbiprofen, omeprazole, dextromethorphan and midazolam as probe drugs respectively assessing CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A activities was administered orally before surgery, day 1 (D1) and 3 (D3) post-surgery and at discharge. Capillary blood samples were collected 2h after cocktail intake to assess metabolic ratios (MR). Serum inflammatory markers (CRP, IL-6, IL-1β, TNF-α, IFN-γ) were also measured in blood. CYP1A2 MR decreased by 53% (p<0.0001) between baseline and the nadir at D1. CYP2C19 and CYP3A activities (MRs) decreased by 57% (p=0.0002) and 61% (p<0.0001) respectively, with the nadir at D3. CYP2B6 and CYP2C9 MRs increased by 120% (p<0.0001) and 79% (p=0.018) respectively and peaked at D1. Surgery did not have a significant impact on CYP2D6 MR. Hip surgery was a good acute inflammation model as CRP, IL-6 and TNF-α peak levels were reached between D1 and day 2 (D2). Acute inflammation modulated CYP activity in an isoform-specific manner, with different magnitudes and kinetics. Acute inflammation may thus have a clinically relevant impact on the pharmacokinetics of these CYP substrates.