The objectives of this experiment were to evaluate and compare underivatized (UND) and precolumn derivatized (DER) methods for quantification of bovine plasma AA by isotope dilution ratio via liquid chromatography-electrospray ionization (ESI)-single quadrupole mass spectrometry. Linearity of the mass-to-charge ratio signal and area signal sensitivity of 12C were evaluated for each AA with 5-point standard curves (range: 1.1-500 µM). Plasma from lactating dairy cows was isolated by centrifugation and deproteinized using 1 N perchloric acid with a final concentration of 0.5 N. Deproteinized plasma was filtered and injected into a 50 × 2-mm column (Imtakt) or extracted, derivatized, and injected into a 250 × 3-mm column (EZ:faast, Phenomenex) and analyzed via liquid chromatography-ESI-single quadrupole mass spectrometry. Coefficients of variation and recovery rates were evaluated using 4 replicates of pooled plasma samples spiked with each AA at concentrations of 10, 20, and 50 µM. In addition, a subset of 24 plasma samples was used to directly compare methods using linear regression, correlation coefficient (r), concordance correlation coefficient (CCC), and Bland-Altman plot test. Both methods showed linearity within the dynamic range analyzed for all essential AA (coefficient of determination, R2 ≥ 0.995) and most other AA, although the UND samples had poor linearity (R2 ≤ 0.990) or peak resolution problems for Asp, Gly, Tyr, and Ser. Moreover, area signal sensitivity for 12C AA was greater for DER samples than for UND samples [range: 2.2× (Pro) to 309.5× (Ala)]. Both methods had recovery rates ranging from 85.7 to 119.8.0%, and none differed from 100% except Gln [20 µM (85.7%) and 50 µM (87.6%)] and Val [50 µM (119.8%)] using the UND method. The UND method had a coefficient of variation ranging from 0.9% (Val) to 7.8% (His), whereas for the DER method the range was 2.2% (Glu) to 8.8% (Asp). The highest correlation coefficient (>0.90) and CCC (>0.90) were observed for Arg, Ile, Leu, Met, Thr, Trp, Val, and Gln, with the Bland-Altman plot test showing minimal mean bias for these AA. Lowest values were observed for His (r = 0.46; CCC = 0.45), Lys (r = 0.76; CCC = 0.75), Ala (r = 0.83; CCC = 0.73), and Glu (r = 0.65; CCC = 0.42). The UND method showed linearity, precision, and accurate recovery rates for most AA, with most essential AA having comparable values between the 2 methods. However, the DER method had greater 12C AA area signal sensitivity, linearity, and recovery rates.