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Following these criterions relative standard deviation less then 15% and relative bias less then 15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences. Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for ve