Background: Vancomycin is a commonly used antibiotic, which requires therapeutic drug monitoring to ensure optimal treatment. Microsampling assays are attractive tools for pediatric clinical research and therapeutic drug monitoring. Results: A LC–MS/MS method for the quantification of vancomycin in human whole blood employing volumetric absorptive microsampling (VAMS^®) devices (20 μl) was developed and validated. Vancomycin was stable in human whole blood VAMS under assay conditions. Stability for vancomycin was established for at least 160 days as dried microsamples at -78°C. Conclusion: This method is currently being utilized for the quantitation of vancomycin in whole blood VAMS for an ongoing pediatric clinical study and representative clinical data are reported.