Background: Vancomycin is usually administered after the dialysis sessions to patients undergoing hemodialysis. Administration of vancomycin during (as opposed to after) sessions would save time, and would be more acceptable to patients and staff, but may lead to vancomycin underexposure. The aim of this study was to propose a new dosing regimen of vancomycin taking into account the dialysis-related losses of vancomycin when administered during dialysis. Methods: In this monocentric prospective study, vancomycin was infused to dialyzed patients during the last hour of the dialysis session at increased doses. Monitoring of vancomycin was performed using repeated blood samples by pharmacokinetics modeling. Patients were treated according to our protocol and guidelines. Results: Twenty patients were included. Vancomycin protocol was efficient: 17 of 20 (85%) patients were cured, 1 needed additional vancomycin treatment, and 2 died (sepsis n = 1, multiple organ failure n = 1). Median pre-dialysis concentration was adequate (16.2 [10.2-24.4] µg/mL), and there was no emergence of resistant bacteria. Infusion of vancomycin during dialysis therefore decreased the exposure to vancomycin by 25% compared to infusion of vancomycin after dialysis. For a typical patient, the dose of vancomycin to be administered during dialysis would be 1.4 g. Conclusion: Administration of vancomycin during the last hour of dialysis session is safe, efficacious with regards to infection control, achieved recommended vancomycin concentrations despite the use of high-flux membranes, and improved patients' quality of life.