Simple and specific analytical methods were developed and validated to quantify vancomycin in plasma and lung tissue, which were obtained from a model of pneumonia in mechanically ventilated piglets. Tissue and plasma samples were precipitated and centrifuged; 100L of the supernatant were injected into the chromatographic system. Ceftazidime was used as the internal standard. The stationary phase was a silica based column Symmetry300C18 (1504.6mm) with pre-column. The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 75mM sodium acetate buffer (pH=3) with (B) acetonitrile (92%/8%;v/v). Isocratic flow rate was set at 0.8mL/min and 0.7mL/min for plasma and tissue samples, respectively. UV absorbance detection was set at 230nm. Standard curves showed good linearity for plasma and pulmonary tissue (r 2>0.99). The lower limits of quantitation were 1.56g/mL and 3.13g/mL for plasma and tissue, respectively. The intra- and inter-day precisions and accuracy all satisfied the acceptance criteria. Both HPLC assays to quantify vancomycin in plasma and pulmonary tissue are rapid, simple, and inexpensive. These methods could be helpful to develop further pharmacokinetic studies of vancomycin penetration in pulmonary tissue. © 2012 Copyright Taylor and Francis Group, LLC.