Background: Accurate analysis of ionized calcium (iCa) is critical for the detection of hypocalcemia or subclinical hypocalcemia. The Edan i15 Vet (EDAN) blood gas device has not been validated for iCa in dairy cows. Objectives:We aimed to validate the EDAN blood gas device against the Gem Premier 3000 (GEM) analyzer by measuring iCa concentrations and evaluating the ability of these measurements, compared with serum total calcium (TCa) concentrations, to diagnose subclinical hypocalcemia.Methods: iCa concentrations were measured with the EDAN and GEM devices, and serum TCa concentrations were measured with a wet biochemistry method with blood from 125 lactating Holstein cows between calving to day 27 postpartum.Results: Bland-Altman plots showed a mean and total bias of 0.05 and 0.24 mmol/L for the EDAN device, respectively. The intercept did not include zero, but the slope included 1.0 in the Passing-Bablok regression. The sensitivity and specificity (Se/Sp) of the EDAN device were 93/94%, 93/90%, 91/93%, and 85/95% for iCa cut-off values of <1.00, 1.05, 1.10, and 1.15 mmol/L, respectively, as determined with the GEM device. The Se/Sp were 57/82% and 72/80% for EDAN and 57/80% and 72/79% for GEM at serum TCa cut-off points <2.15 and <2.00 mmol/L, respectively. The average iCa concentrations analyzed with the GEM and EDAN devices were 1.04 ± 0.18 and 1.09 ± 0.17, respectively. Conclusions:The EDAN device did not have satisfactory agreement with GEM and could not be used interchangeably, but it had satisfactory Se/Sp to diagnose subclinical hypocalcemia compared with the GEM-derived iCa cut-off points. Serum TCa concentration cut-off values were not suitable for diagnosing subclinical hypocalcemia because of unsatisfactory Se/Sp compared with iCa concentrations analyzed by the GEM and EDAN devices. The iCa values analyzed using the EDAN and GEM devices were consistent with previously reported data.