Application of nitrofuran antimicrobials at food production animals was prohibited by Commission Regulation 2003/181/EC because of their potential carcinogenic and mutagenic effects on humans. Main protein-bound metabolites of nitofurans are 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD), semicarbazide (SEM) and 3-amino-2-oxazolidinone (AOZ). Since then numerous costly liquid chromatography with tandem mass spectrometry (LC/MS/MS) methods have been developed for screening and confirmation of nitrofuran metabolites in line with the EU requirements for performing official controls. As an inexpensive and less time consuming alternative, enzyme-immunoassay methods were developed for screening of the respective compounds. In this study validation and evaluation of four commercial enzyme-linked immunosorbent assay (ELISA) has been performed. According to the requirements of Commission Decision 2002/657/EC, different performance characteristics (specificity, detection capability, precision) for various matrices (liver, eggs, honey) have been determined for each kit. The validation study has confirmed that the methods studied possess suitable characteristics: detection limits between 0.126 and 0.240 μg/kg, detection capabilities ≤1.0 μg/kg and the inter-day precision in the range from 16.20% to 22.11 %. The validation study was finalized by participation in FAPAS Proficiency testing scheme in 2011, and the obtained results have confirmed the capability of applied methods for unambiguous discrimination between negative and positive sample.