Supplementation of choline in dairy cattle decreases liver lipid accumulation, potentially through increased very low density lipoprotein (VLDL) export. The direct effect of choline supplementation on VLDL export has not been confirmed due to an inability to quantify bovine VLDL. In other species, VLDL is separated from low- and high-density lipoproteins based on size; however, bovine VLDL differs in density compared with nonruminant VLDL particles due to lipid profile. The objectives of this experiment were to validate a bovine-specific ELISA technique for use in quantification of bovine VLDL and determine the effect of increasing concentrations of choline chloride (CC) and dL-methionine (dLM) during fatty acid challenge on VLDL export in cell culture. Primary hepatocytes isolated from 4 Holstein calves were maintained as monolayer cultures for 24 h before treatment with CC (33, 100, 2000, 4500 µM) and dLM (16, 30, 100, 300 µM) within a factorial design, with 1 mM FA cocktail. Treatments mimicked expected physiological concentrations. After 24 h, medium was collected for VLDL quantification by ELISA. The ELISA (NeoBiolab, Cambridge, MA) is a competitive immunoassay that utilizes antibodies for apolipoprotein B100 and C, which in combination uniquely binds to VLDL particles. The assay was validated using cell culture and postpartum bovine serum samples with purified LDL and HDL spikes. Recovery of samples with spiked standards, compared with independently analyzed samples and standards, was 102 ± 1%. Cell culture data were analyzed using PROC MIXED of SAS 9.4 with linear and quadratic contrasts including fixed effect of treatment and random effect of calf. Interactions were not significant and therefore only main effects are discussed. Increasing concentrations of CC linearly increased (P = 0.02) VLDL export from hepatocytes. Treatment with dLM did not alter (P = 0.7) VLDL export. This research validates an ELISA for use in quantification of bovine VLDL. Furthermore, these data support the role of CC, but not dLM, supplementation in increasing VLDL export from hepatocytes.