The objectives of this study were (1) to determine the differences in IgG and total protein (TP) content of serum and plasma samples collected from the same calves; (2) to evaluate the correlation between calf serum and plasma IgG levels, Brix scores, and TP concentrations; (3) to determine whether different cut-off values should be used for plasma and serum to assess failure of transfer of passive immunity (FTPI) in dairy calves; and (4) to evaluate the level of agreement between results obtained from using serum and plasma samples of the same calves to assess FTPI using optimal cut-off values. Blood samples (n = 217) were collected from Holstein calves at 3 to 10 d of age on 30 commercial dairy farms in Nova Scotia and Newfoundland, Canada. Paired serum and plasma samples were analyzed for IgG concentration by the reference radial immunodiffusion assay, transmission infrared (TIR) spectroscopy, digital and optical Brix refractometers, and optical TP refractometer. The IgG concentrations measured by RID and TIR spectroscopy in serum were similar to those in plasma. However, the Brix and TP refractometer readings were significantly higher in plasma than in serum. The prevalence of FTPI in serum and plasma samples based on a RID-IgG concentration <10 g/L was 43.3 and 46.5%, respectively. The RID-IgG concentration was correlated with TIR-IgG (r = 0.92 and 0.89), digital Brix (r = 0.80 and 0.80), optical Brix (r = 0.77 and 0.77), and optical TP (r = 0.75 and 0.77) refractometers in serum and plasma, respectively. The correlations between paired serum and plasma IgG content were 0.85 by TIR spectroscopy, 0.80 by digital Brix, 0.77 by optical Brix, and 0.79 by optical TP refractometer. The optimal cut-off values for TIR spectroscopy, digital Brix, optical Brix, and TP refractometers to assess FTPI using serum were 13.1 g/L, 8.7% Brix, 8.4% Brix and 5.1 g/dL, respectively; and the optimal cut-off values with plasma were 13.4 g/L, 9.4% Brix, 9.3% Brix and 5.8 g/dL, respectively. When using these optimal cut-off values, the level of agreement (88.1%) between results derived from testing serum and plasma by TIR spectroscopy was substantial, with a kappa (κ) value of 0.76. The results derived from testing serum and plasma by digital Brix refractometer showed substantial agreement (83.4%), with a κ value of 0.65, which is higher than the agreement and κ value (74.7% and 0.51) reported for the optical Brix refractometer. Substantial agreement (81.6%) between serum and plasma TP was also obtained when using the optical TP refractometer, with a κ value of 0.63. In conclusion, serum or plasma samples can be used interchangeably for measuring IgG concentrations and assessing FTPI in dairy calves. However, different cut-offs must be used to assess FTPI depending on the sample matrix. Furthermore, results obtained from serum samples showed higher agreement with the reference RID assay than those obtained from plasma samples.