Traditional polymorphonuclear (PMN) leukocyte (neutrophil) function assays of oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. OB is evaluated by measuring PMN median fluorescence intensity (MFI) after in vitro stimulation. PC is measured by engulfment of fluorescent beads by PMN. DQ^TM ovalbumin (DQ-OVA) labels a pH-insensitive compound that fluoresces upon proteolytic degradation after endocytosis. DQ-OVA might, therefore, be informative about an additional pathway of pathogen handling by PMN. This study evaluated the use of the DQ-OVA assay for the assessment of PMN function, and the associations among OB, PC, and DQ-OVA outcomes in PMN isolated from blood of dairy cows between 5 and 21 d postpartum. The PMN function outcomes were assessed with mixed linear regression models. For the validation assay (9 cows in 3 replicates), PMN cocultured with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D and compared with control non-stimulated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units, P = 0.4) and lesser (593 ± 55 units, P = 0.03) MFI relative to non-stimulated PMN (791 ± 154 units), respectively, indicating that DQ-OVA reflected enhanced or reduced endocytic and proteolytic function. From 160 samples from 40 cows, the only significant association among PMN assays was between DQ-OVA MFI and PC (Pearson r = 0.16; P = 0.04). When values of MFI were categorized according to the first, second and third, or fourth quartiles, agreement beyond chance (κ statistic) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays since it provides additional information regarding antigen processing, but it is not a substitute for either.