A surface plasmon resonance (SPR) method is described for quantitative determination of α-lactalbumin, β-lactoglobulin, bovine serum albumin, lactoferrin and immunoglobulin G simultaneously in raw and processed milk samples, whey fractions and various milk-derived products, with six samples per assay. Immobilized antibody stability and reproducibility of analyses were studied over time for 25 independent runs (n = 150), giving a maximum relative standard deviation of <4%. Immobilized antibodies showed negligible non-specific interactions (<2–4 response units (RU)) and negligible cross-reactivity towards other milk components (<1 RU). Regeneration of immobilized antibodies with glycine at pH 1.75 was shown to be optimal for maintaining the SPR response between samples. The method compared well with reverse-phase liquid chromatography and standard enzyme-linked immunosorbent assays. Improved quantitation of samples containing both high and low abundance proteins was achieved by injecting each sample set twice, using different dilutions to match protein concentrations to their optimal calibration ranges.