Eight cattle (288 ± 22 kg) were treated with 2.2 mg/kg of body weight of flunixin free acid in a crossover design by subcutaneous (SC) and intravenous (IV) administration. After a minimum 1:10 dilution with 50 mM phosphate buffer, a commercial immunoassay was adapted to determine plasma concentrations of flunixin. The limit of detection was 0.42 ng/mL and the working range was 0.76-66.4 ng/mL when adjusted with the dilution factor. Plasma samples were extracted using mixed-mode cation exchange solid phase extraction prior to the LC-MS/MS analyses. The linear calibration curve for LC-MS/MS was 0.5-2000 ng/mL with a limit of detection of 0.1 ng/mL for flunixin and 0.3 ng/mL for 5-hydroxy flunixin. Flunixin concentrations determined using the ELISAs were compared to concentrations derived from the same samples using LC-MS/MS analyses. Pharmacokinetic parameters of time versus concentration data from each analysis were estimated and compared. Differences (P < 0.05) in estimates of area under the curve, volume of distribution, and clearance were apparent between ELISA and LC-MS/MS analyses after IV dosing; after SC dosing, however, there were no differences among the estimated parameters between the two methods. Quantitative immunoassay was a satisfactory method of flunixin analysis and that it would be difficult to differentiate routes of administration in healthy beef cattle based on the plasma elimination profile of flunixin after IV or SC administration.