In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20β-dihydroprogesterone (20β-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3β-dihydroprogesterone (3β-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5β-reduced progestogens (5β-dihydroprogesterone (5β-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20β-DHP. Further studies will need to focus particularly on the period around luteolysis. •We monitored 12 progestogens in plasma of cyclic cattle using a novel LC-MS based approach.•We detected 11 differentially abundant progestogens in the plasma of cyclic cattle.•To the best of our knowledge, we were the first to detect the presence of 3α-DHP and epipregnanolone within bovine plasma.•The profiles of the P4 metabolites widely followed the profile of their precursor P4, but were distinct around luteolysis.