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Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze–thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.