Neonicotinoids have become the most widely used insecticides in world with rapid growth in applications as seed coatings. Nontarget organisms are exposed to concentrated levels of pesticidal active ingredients through ingestion of treated seeds. To better understand pesticide fate, analytical methods are necessary to rapidly screen and accurately quantitate contaminants in environmental and biological matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly employed for neonicotinoid analyses but requires expensive analytical instrumentation and potentially laborious sample preparation. Enzyme-linked immunosorbent assays (ELISAs) are efficient and sensitive alternative methods for neonicotinoid analyses, that does not require costly equipment. In this study, application of ELISA was compared to LC-MS/MS in the quantitation of imidacloprid in plasma, liver, and fecal matter from Japanese quail (Coturnix japonica) orally exposed to imidacloprid-treated wheat seeds. Two major imidacloprid metabolites, 5-OH-imidacloprid and imidacloprid-olefin, were found to cross-react within the immunoassay, confounding results. Imidacloprid concentrations from ELISA analysis ranged from 90.8-181 ng/mL, 7.90-40.4 ng/g, and 989-4404 ng/g in plasma, liver, and fecal matter, respectively. In comparison, LC-MS/MS imidacloprid concentrations ranged from 32.8-188 ng/mL for plasma, 18.7-116 ng/g for liver, and 51.3-427 ng/g for fecal matter. Imidacloprid concentrations determined by ELISA were generally higher in plasma and exceptionally higher in fecal matter compared to LC-MS/MS, Liver imidacloprid concentrations were higher when analyzed by LC-MS/MS. Good correlation was observed between imidacloprid concentrations determined in plasma (r2 = 0.93) and fecal matter (r2 = 0.89) by ELISA and LC-MS/MS, whereas a poor correlation (r2 = 0.35) was found in liver. In the analysis of these biological matrices, ELISA was found to be a good screening tool to measure active ingredients along with their metabolites. However, accurate quantitation should be completed with chromatographic based techniques such as LC-MS/MS.