Pregnancy associated glycoproteins (PAG) can be used as a biomarker for early pregnancy diagnosis, so accurate and consistent PAG detection is critical. The objective of this study was to determine if plasma and serum PAG concentrations were altered when centrifugation occurred at different times post-collection, when subjected to repeated freezing and thawing, and when monoclonal antibodies were kept in frequently or infrequently opened containers. Plasma (n = 4) and serum (n = 4) samples were collected from two open cows and two pregnant cows 28 days after artificial insemination. Pregnancy status was determined via transrectal ultrasonography. Plasma and serum samples were evenly separated and either centrifuged on the day of collection, or placed at 4°C and centrifuged the next day. An in-house PAG ELISA was performed on all samples before freezing (NOTHAW), after being frozen for one week (INTACT), after one freeze/thaw cycle (THAW1), two freeze/thaw cycles (THAW2), and three freeze/thaw cycles (THAW3). Data were analyzed using one-way ANOVA (GLM procedure, SAS 9.4). All samples from open cows were below the baseline of the assay. For pregnant cows, plasma samples had greater PAG concentrations than serum samples (11.84 vs 3.30 ± 0.66 ng/mL, respectively, P > 0.05). No differences were observed for day of centrifugation in both plasma and serum samples (P = 0.50 and P = 0.60, respectively) and in handling of monoclonal antibodies (P = 0.90). Freezing and thawing did not impact PAG concentrations in plasma samples (P = 0.19), but did alter serum concentrations (P = 0.01). Specifically, THAW1 (1.98 ng/mL) and THAW2 (1.42 ng/mL) serum PAG concentrations were lower compared to NOTHAW, THAW3, and INTACT samples (4.66, 4.85, and 3.57 ng/mL, respectively). Based on these data, plasma yields more consistent results than serum, even after several freeze-thaw cycles, and handling of monoclonal antibodies or time of centrifugation has no significant effect on measured PAG.