Inadequate tissue penetration of antibiotics can lead to therapeutic failure and bacterial resistance. Pharmacokinetic evaluation of antibiotics should therefore be based on tissue rather than serum concentrations. Over several years, tissue concentration data obtained by methods such as tissue biopsies have flawed the correct interpretation of antibiotic tissue distribution. Microdialysis--a semi-invasive catheter-based sampling technique--has been employed for the in vivo measurement of antibiotic tissue pharmacokinetics. Owing to selective access to the target site for most anti-infective drugs, microdialysis satisfies regulatory requirements for pharmacokinetic distribution studies and might become a reference technique for tissue distribution studies in the near future. Furthermore, microdialysis might contribute to the definition of meaningful surrogate markers for antibiotic efficiency during drug development.