The optimal conditions for determining the concentration of progesterone in the blood plasma of cattle were selected by high-performance liquid chromatography-tandem mass spectrometry, and the relationship of the level of this progestin with fertilization after treatment of cows with hormonal preparations was studied. It was found that the time of progesterone peak release under the selected chromatography mode is within 2.7 minutes, and the optimal precursor ion is [M+H]+ m/z=315.3. The developed technique makes it possible to determine the endogenous level of the steroid in the blood of cattle with high accuracy (up to hundredths of ng/ml). Analysis of progesterone concentration in cows at the time of the final injection of the prostaglandin analogue showed that animals with different reproductive status have no significant differences in the content of this steroid. The most statistically significant indicators were obtained on the day of insemination. Thus, in infertile cows, the hormone concentration was 1.9...4.2 times lower than in fertilized cows (P<0.05). The higher steroid content in fertilized animals at the time of insemination most likely reflects the ovarian response to the preovulatory release of luteinizing hormone and the readiness of the follicle for ovulation. Keywords: cattle; high-performance liquid chromatomass spectrometry; progesterone; synchronization of sexual cyclicity; fertilization; infertility.