The differences between lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response using luminol-amplified chemiluminescence (CL) are currently unknown in cattle. Luminol-dependent CL measures the amount of reactive oxygen species released from leukocytes after stimulation with LPS and/or PMA. The objective of this study was to compare in vitro the effect of PMA and LPS on oxidative response in whole blood of dairy cows during lactation as a rapid means of assaying the oxidative response of blood leukocytes. Jugular blood (20 mL) was collected from 6 healthy multiparous Holstein dairy cows in mid-lactation (>90 DIM) using EDTA Vacutainer tubes. For each cow, 500 µL of blood was incubated at final concentrations of either 0, 200, 800 or 1,600 ng/mL of PMA or LPS for 15 min at 37°C using a heating block. After incubation, oxidative response of whole blood was measured using a chemiluminometer. Data were analyzed by ANOVA using the PROC MIXED procedure of SAS. Overall, whole blood incubated with PMA resulted in higher (P < 0.001) CL values (800 ng/mL; 2635 relative units) than LPS (800 ng/mL; 777 relative units). In PMA, a significant dose response relationship was observed where incubation with 200, 800 or 1,600 ng/mL resulted in progressively higher CL values than 0 ng/mL. In addition, incubation with PMA resulted in a higher CL values when compared with LPS. In conclusion, although both LPS and PMA both generated an oxidative response measurable by CL, PMA elicited a CL response greater than that of LPS. The data suggest that PMA stimulation of cells in whole blood may serve as a rapid test of oxidative burst responsiveness to assess a vital aspect of immune function in dairy cows.