Optimal use of antibiotics is a key component of the management of sepsis. The purpose of this study was to develop a modification of the time-to-positivity (T(pos)) assay for use in the acute management of septic patients. Initial laboratory experiments, followed by ex-vivo validation and pilot studies, were performed with a small number of healthy human volunteers and 46 septic patients on a general intensive care unit, chosen on the basis of their antibiotic regimen. The study demonstrated that the T(pos) assay could be used as a surrogate for antimicrobial activity, and provided preliminary data to demonstrate how this approach might be used to monitor the efficacy of antibiotic therapy in septic patients. The T(pos) assay might offer a quick and convenient way to improve the efficacy of antibiotic therapy in septic patients, and further prospective large-scale studies are now warranted.