d Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic (PK)/pharmacodynamic (PD) studies in situations where venous blood sampling is logistically difficult. We sought to develop, validate, and apply a DBS assay for rifampin (RIF), fusidic acid (FUS), and ciprofloxacin (CIP). These antibiotics are considered active against organisms in biofilms and are therefore commonly used for the treatment of infections associated with prosthetic implants. A liquid chromatography-mass spectroscopy DBS assay was developed and validated, including red cell partitioning and thermal stability for each drug and the rifampin metabolite desacetyl rifampin (Des-RIF). Plasma and DBS concentrations in 10 healthy adults were compared, and the concentration-time profiles were incorporated into population PK models. The limits of quantification for RIF, Des-RIF, CIP, and FUS in DBS were 15 g/liter, 14 g/liter, 25 g/liter, and 153 g/liter, respectively. Adjusting for hematocrit, red cell partitioning, and relative recovery, DBS-predicted plasma concentrations were comparable to measured plasma concentrations for each antibiotic (r > 0.95; P < 0.0001), and Bland-Altman plots showed no significant bias. The final population PK estimates of clearance, volume of distribution, and time above threshold MICs for measured and DBS-predicted plasma concentrations were comparable. These drugs were stable in DBSs for at least 10 days at room temperature and 1 month at 4°C. The present DBS antibiotic assays are robust and can be used as surrogates for plasma concentrations to provide valid PK and PK/PD data in a variety of clinical situations, including therapeutic drug monitoring or studies of implant infections. P harmacokinetic (PK)/pharmacodynamic (PD) studies of infectious diseases explore the triangular relationship between antibiotic exposure, the antibiotic susceptibility of the infecting organism (taken as the MIC), and predefined clinical outcomes (1). This allows estimation of microbiological susceptibility "breakpoints," facilitates the design of optimal dosing regimens (2), and provides data relating to the emergence of drug-resistant organisms (3, 4). Most PK/PD studies are performed in animal models or in highly selected samples of adults, such as patients in intensive care units, for whom sequential sampling of sufficient volumes of venous blood is both feasible and ethical. In contrast, PK/PD studies are rarely performed on other hospitalized or ambulatory patient populations due to difficulties associated with blood sampling. Relevant examples include patients receiving treatment for implant infections, young children, and patients treated in nonurban or resource-poor settings.Measurement of drug concentrations in dried blood spots (DBSs) represents a new approach to overcome the limitations of traditional therapeutic drug monitoring and PK/PD studies. DBSs are a convenient and inexpensive means of sampling and storage of whole blood for subsequent assays. Low-volume finger prick samples (10 to 20 l) are taken on f...