An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard (0.15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg), for the kit were calculated.