Microdialysis is a technique used to measure the unbound antibiotic concentration in the interstitial spaces, the target site of action. In vitro recovery studies are essential to calibrating the microdialysis system for in vivo studies. The effect of a combination of antibiotics on recovery into microdialysate requires investigation. In vitro microdialysis recovery studies were conducted on a combination of vancomycin and tobramycin, in a simulated in vivo model. Comparison was made between recoveries for three different concentrations and three different perfusate flow rates. The overall relative recovery for vancomycin was lower than that of tobramycin. For tobramycin, a concentration of 20μg/mL and flow rate of 1.0μL/min had the best recovery. A concentration of 5.0μg/mL and flow rate of 1.0μL/min yielded maximal recovery for vancomycin. Large molecular size and higher protein binding resulted in lower relative recoveries for vancomycin. Perfusate flow rates and drug concentrations affected the relative recovery when a combination of vancomycin and tobramycin was tested. Low perfusate flow rates were associated with higher recovery rates. For combination antibiotic measurement which includes agents that are highly protein bound, in vitro studies performed prior to in vivo studies may ensure the reliable measurement of unbound concentrations.