Milk processing industries and distributors have problems with adulteration of liquid milk by the addition of bovine cheese whey. Recently, the detection of fraudulent manipulation of milk with whey has focused on the identification of glycomacropeptide (GMP). Current non-immunological methods to detect GMP in dairy products are expensive and time-consuming or have low sensitivity. In this study, a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of whey in raw milk was developed, using a polyclonal rabbit anti-GMP antibody. Calibration curves were constructed by analyzing raw milk standards containing different known concentrations of liquid cheese whey (0.02–20%). The method had a detection limit of 0.047% ( v/v ) and a quantification limit of 0.14% ( v/v ). The antibody showed high specificity and no cross-reaction with milk components (other than κ-casein) and was successful in detecting GMP in dairy commercial products. The recovery ratio was between 95.62% and 113.88% for all matrices tested. The intra-assay and interassay coefficients of variation were <6% and <7%, respectively. Finally, it can be stored for 3 months in the form of a ready-to-use kit, while maintaining its accuracy and reproducibility.