In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)-LC–MS/MS method for the simultaneous quantification of four antibiotics: piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10 μL human blood. The novel VAMS-LC–MS/MS method has been compared with a dried blood spot (DBS)-based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step at 37 °C for 10 min. LC–MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC–MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics.