Serum or plasma concentrations of nonesterified fatty acids (NEFA) are commonly used as biomarkers of lipolysis during the periparturient period in dairy cows. However, NEFA quantification usually requires sample submission to specialized diagnostic laboratories, at significant cost. Alternative methods for the measurement of NEFA concentrations are needed that decrease the cost per sample without compromising accuracy and precision. Our study compared the quantification of NEFA between the gold standard diagnostic laboratory method and 2 alternative methods: a 96-well plate protocol and a small-scale chemistry analyzer (CataChemWell-T; Catachem Inc., Oxford, CT). We used a total of 147 plasma samples collected from cows 7 to 13 d before their expected calving date (7 ± 3.3; mean ± SD days before actual calving) were used. We used linear and Passing–Bablok regression to identify systematic and proportional bias between the alternative methods and the gold standard. We also examined the level of agreement between each alternative method and the gold standard using Bland–Altman plots. We calculated the sensitivity and specificity of the alternative methods for detecting animals with excessive lipid mobilization prepartum (defined as NEFA concentration ≥0.30 mM by the gold standard test). We identified a constant difference between each of the alternative NEFA determination methods and the gold standard. Nevertheless, the mean bias was relatively small (–0.03 mM and –0.02 mM for the 96-well plate and small-scale analyzer methods, respectively). However, this tendency to underestimate NEFA concentrations had only a minimal effect on the ability of the tests to detect cows with excessive lipid mobilization prepartum (specificity 100%; sensitivity 88.9 and 94.4% for the 96-well plate and small-scale analyzer methods, respectively). The 96-well plate and small-scale chemistry analyzer methods tested in this study are suitable for the quantification of NEFA concentrations in plasma and the dichotomous classification of samples as indicators of excessive prepartum lipid mobilization.