Pilot studies of the pharmacokinetics of Gestobutanoil (GB) showed that it undergoes rapid biotransformation forming two metabolites, which quantitative analysis was significant. An HPLC-MS technique was developed for the simultaneous quantification of GB and its two metabolites in rat serum. Due to the nature of GB and one of its metabolites, the ESI-MS-detection was ineffective. The ionization problem was solved with APCI. The detection of GB and one of its metabolites was carried out by the fragments of their molecules formed in the ionization chamber. The second metabolite formed [M+H]⁺ ions. During the method validation following characteristics were checked out: specificity, linearity, precision, accuracy, matrix effect, stability. The limit of quantification for each analyte was 10 ng/ml. Pharmacokinetics studies have shown that biotransformation of GB is so fast that it wasn't detected in rat serum even in 15 minutes after administration. The pharmacokinetics of two metabolites of GB was described.