Surgical site infections are common, so effective antibiotic concentrations at the sites of infection, i.e., in the interstitial fluid (ISF), are required. The aim of this study was to evaluate contemporary perioperative prophylactic dosing of cefazolin by determining plasma and subcutaneous ISF concentrations in patients undergoing elective/semielective abdominal aortic aneurysm (AAA) open repair surgery. This was a prospective pharmacokinetic study in a tertiary referral hospital. Cefazolin (2 g) was administered as a 3-min slow bolus 30 min prior to incision in 12 enrolled patients undergoing elective/semielective AAA open repair surgery. Serial blood, urine, and ISF (via microdialysis) samples were collected and analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Cardiac output was determined using pulse waveform contours with Vigileo. The recruited patients had a median (interquartile range) age of 70 (66 to 76) years and weight of 88 (81 to 95) kg. The median (interquartile range) terminal volume of distribution was 0.14 (0.11 to 0.15) liter/kg, total clearance was 0.05 (0.03 to 0.06) liter/h, and minimum observed unbound concentration was 5.7 (5.4 to 8.1) mg/liter. The penetration of unbound drug from plasma to ISF was 85% (78% to 106%). We found correlations present, albeit weak, between cefazolin clearance and cardiac output (r(2) = 0.11) and urinary creatinine clearance (r(2) = 0.12). In conclusion, we found that a single 2-g dose of cefazolin administered 30 min before incision provides plasma and ISF concentrations in excess of the likely MICs for susceptible pathogens in patients undergoing AAA open repair surgery.