During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC(50) (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 +/- 11.0%) and commercially available bovine (20.2 +/- 8.3%) or human serum albumin (56.9 +/- 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 +/- 11.6%; bovine serum albumin, 12.4 +/- 4.8%; human serum albumin, 17.8 +/- 11.5%). The MICs and EC(50)s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC(50)s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values.