The purpose of this study was to develop and validate an in-house competitive enzyme immune assay (hEIA) to measure the amount of progesterone (P4) in the serum of dairy cattle. Blood samples were collected from randomly selected multiparous Holstein cows at predetermined times of their estrous cycle (d −5 to 12), and serum separated for analysis. The in-house assay was then compared with a commercial EIA kit (cEIA; Ovucheck Plasma TRM546, Biovet Inc., QC, Canada) and a RIA method validated by Pohler et al. (2016). In short, the protocol used a double-antibody (secondary antibody, Goat antimouse; and a primary antibody, monoclonal progesterone) and matching enzyme conjugate label (Progesterone-3-HRP). 96 well microtiter plates were coated with the secondary antibody, incubated then later blocked using the primary antibody. Progesterone standards ranged from 0.1 to 20 ng/mL. Progesterone was extracted from samples using petroleum ether. Quality controls were created from serum stripped of steroids using a charcoal procedure. Microplate reader wavelength was set at 450nm and P4 calculated using a Log-Logit regression line. Both low (0.45 ng/mL) and high (6.43 ng/mL) concentration internal controls were run on 6 plates to calculate intraplate and interplate %CVs. The intraplate CV was 7.68% and 5.14%, and the interplate was 12.5 and 11.0%, respectively for the low and high samples. The prepared quality controls (2.5 ng/mL) were tested on the same 6 plates and resulted in a 6.56 and 3.61%CV for intra and interplate variability. When compared with the other two validated assays, the correlations among the 3 assays were statistically significant (P < 0.01) and yielded the following correlations: h-EIA and RIA (r = 0.95; slope=1.29); c-EIA and RIA (r = 0.91; slope = 1.51); hEIA and cEIA (r = 0.96; slope = 1.10). When agreement tests were performed to detect samples below and above 1 ng/mL, the kappa values were 1.00 for h-EIA and RIA; 0.78 for c-EIA and RIA; and 0.78 for hEIA and cEIA. In conclusion, the in-house EIA method is a reliable assay to accurately measure concentrations of P4 in bovine serum that is suitable for commercial and research purposes.