Introduction: Cefazolin is the first choice antibiotic prophylaxis during cardiac surgery and is widely used to prevent deep sternal wound infections (DSWI). Recently, the Dutch guideline has been changed where the cefazolin dose given at the onset of cardiopulmonary bypass (CPB), has been removed and the postoperative dosages are both halved. The aim of the study is to assess if adequate cefazolin plasma levels are obtained with the new Dutch guideline. Methods: Twenty-four adults undergoing cardiac surgery with cardiopulmonary bypass (CPB), receiving cefazolin were studied. The main goal is 100% fT >MIC during surgery and epidemiological cutoff is 2 mg/L. During the postoperative phase the goal is 40% fT >MIC. Per patient 9 to 11 blood samples were collected to measure plasma cefazolin concentrations. Results: During surgery 100% of the measured concentrations were above the ECOFF of 2mg/L. BMI was not significantly associated with unbound cefazolin concentrations. Sex, age, albumin levels preoperative and duration of CPB were also not significantly associated with cefazolin unbound concentration. Redose was significantly associated with higher total plasma concentration. In the postoperative phase 85% were above the ECOFF of 2mg/L. Conclusion: This study shows that the unbound cefazolin concentration during surgery is 100% above the ECOFF of 2mg/L, though sometimes close to the minimum. In the postoperative period more than 85% was above the ECOFF of 2mg/L.