Background: Several factors may influence the concentration of serum globulins in healthy cows and thereby affect clinical interpretation; however, few studies have addressed sources of variation in globulin values. Objectives: The purpose of this study was to compare colorimetry-based total serum globulin values with electrophoretically-determined serum globulin fractions and with IgG concentration, and to evaluate diurnal and long-term physiological variation and the effects of lactation and venipuncture site on serum globulin concentrations in Holstein dairy cattle. Methods: Serum total globulin and albumin concentrations were analyzed by colorimetry and electrophoresis in 86 lactating cows; IgG concentrations were determined by radial immunodiffusion in 41 dry and 34 lactating cows. Serum globulins were analyzed hourly for 24 hours in 8 lactating cows and weekly for 15 weeks in 6 additional cows. Globulin concentrations were compared in samples obtained from jugular and coccygeal venipuncture sites in 4 cows. Results were analyzed using parametric statistical tests. Results: Colorimetry-based total serum globulin concentrations correlated well with y-globulin fractions (r2= 0.87) and IgG concentrations (r2= 0.91). Diurnal variation of total serum globulins concentration was significant (P= .01); however, globulins did not vary significantly over a 15-week period. Mean serum globulins concentration in samples obtained from the jugular vein was 2.35 g/L higher than that in samples obtained by coccygeal venipuncture (P < .0001). Conclusions: The colorimetric method used widely in routine laboratory analyses remains a useful test for globulins determination in dairy cattle. However, time of sampling and venipuncture site should be considered in the interpretation of serum globulins on serial or interindividual specimens.