Vancomycin (VAN) is an effective antibiotic due to its broad-spectrum bactericidal action. High performance liquid chromatography (HPLC), a powerful analytical technique is used for the in vitro/ in vivo quantification of VAN. The current study was aimed to detect the VAN from in vitro as well as the plasma after the extraction from blood of rabbits. The method was developed and validated according to International Council on Harmonization (ICH) Q2 R1 guidelines. Results showed that the peak of VAN was recorded at 2.96 and 2.57 min, respectively in vitro and serum. The coefficient of VAN turned out to be >0.9994 each for in vitro and in vivo samples. VAN was found linear in the range of 6.2-25000ng/mL. The values of accuracy and precision in terms of coefficient of variation (CV) were less than 2%, indicating the validity of the method. The values for LOD and LOQ were estimated to be 1.5 and 4.5ng/mL, correspondingly, which were lower than the values calculated from in vitro media. Furthermore, the score of the greenness found out to be 0.81, depicting good score using AGREE tool. It was concluded that the developed method was found accurate, precise, robust, rugged, linear, detectable and quantifiable at prepared analytical concentrations and could be used for in vitro and in vivo VAN determination.