The objectives of the study were to compare various methods to determine flunixin in test samples collected periodically from horses after intramuscular (IM) and intravenous (IV) dosing at the maximum recommended dosage and to document detection times for this drug in test samples. Flunixin, a nonsteroidal anti-inflammatory drug approved for use in horses, was administered to eight mares in five consecutive daily doses of 1.1 mg per kilogram of body weight by the IM or IV route. Flunixin was detected in urine samples collected at various times after drug administration by flunixin enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatographic-mass spectrometric (GC-MS) methods. Detection time was defined as the time period over which flunixin was detected and was dependent on the method used. The shortest detection times were 24 to 48 h and were observed when the TLC method was used. On the other hand, detection times were as long as 15 days when HPLC, GC-MS, and flunixin ELISA methods were used. The use of these more sensitive tests to monitor official samples collected from racehorses could result in positive tests for flunixin when it is exerting no detectable clinical effects because it produces clinical effects lasting only 24-36 h in horses.