We previously designed a cyclized nine-amino acid peptide, AFPep, to resist the hydrolysis of exopeptidases. A major finding was that AFPep inhibited estrogen-induced development and growth of experimental breast cancers. However, no detailed pharmacokinetic study has ever been performed to understand the fate and bioavailability of the peptide in animal models. The present study aims to develop a liquid chromatography configured with tandem mass spectrometry (LC-MS/MS) method to quantify the AFPep in mammalian serum. Our method was validated in terms of accuracy, precision, selectivity, sensitivity, stability and reproducibility. The developed method was found to be accurate and precise with a low limit of quantification (LLOQ) and a low limit of detection (LLOD) of 1.96 ng/ml and 0.65 ng/ml, respectively; method selectivity was confirmed by the absence of any matrix interference with the analytic peak. The constructed calibration curve was linear in the range of 2-5,000 ng/ml, with a regression coefficient of 0.998. Average recovery of AFPep from fortified samples (n = 6) was 98% with a relative standard deviation (RSD) of 5.3%. In addition, the peptide was found to be functionally stable in mouse serum at room temperature for 24 hrs. Overall, the established method provides rapid, sensitive, rugged, and robust LC-MS analysis for the quantitative determination of AFPep in biological matrices. This methodology was applied to establish a preliminary pharmacokinetic profile of AFPep following its intravenous administration to mice, dogs and monkeys.