Background Fosamprenavir (FSV) is used for the treatment of HIV infections. It is a prodrug of the protease inhibitor and antiretroviral drug amprenavir. Aim This research work described about the estimation of FSV in spiked human plasma using electrospray ionization, LC-MS/MS technique, and its application to pharmacokinetic study in rabbits. Materials and methods Liquid–liquid extraction technique was used for the extraction of FSV in spiked human plasma. The separation was achieved using ZORBAX SB-C18 column with 4.6 mm internal diameter with 5 μm particle size using acetonitrile: 5 mmol/l ammonium acetate in water (85 : 15, v/v) as a mobile phase. Positive ion mode was selected for the product ion mass spectra, m/z 585.6–418.2 for FSV and m/z 589.2–469.1 for FSV-deuterated (internal standard), The US Food and Drug Administration guidelines were adopted for successful validation of the developed method. Results The retention time of FSV was found to be 1.51 min, for FSV-deuterated it was 1.62 min, with a runtime of 2.5 min. The present method exhibits excellent intraday and interday accuracy with %nominal 95–98.4% and precision percentage coefficient variation up to 3% in all quality control (QC) levels. The developed method demonstrated excellent matrix and analyte selectivity (%interference=0), matrix effect (matrix factor 2.09 at lower quantitation limit and 1.14 at high QC level) and satisfactory stability study results in all types (%nominal 94.03–100.80%). The linearity range was found to be 0.510–200.185 ng/ml with a correlation coefficient (r2) of 0.998. The calculated accuracy and precision values in the ruggedness study were within 15–20% in all QC levels. The percentage coefficient variation of the pharmacokinetic study on rabbit plasma samples was also conducted and the parameters of FSV showed Tmax of 2 h and the mean Cmax, AUC0→t and AUC0→α for test formulation were 98.6, 351.3, and 354.9, respectively. Conclusion This method was successfully optimized, validated, and applied favorable for the pharmacokinetic study of marketed formulation in rabbit blood samples in a single oral human-equivalent dose. The applicability of the developed method undoubtedly can further extend during preclinical and clinical trials.