Current analytical methods for milk proteins lack the capacity to simultaneously separate and quantify the six major bovine milk proteins and their genetic variants. A method is described that simultaneously separates and quantifies the six major bovine milk proteins. The separation is based on reversed-phase partitioning of the six major milk proteins and several genetic variants of kappa-casein, beta-casein, and beta-lactoglobulin. The described method has for each of the six milk proteins a linear quantitative response, precision (coefficient of variation below 5.1% within days of analysis, and below 7.1% between days of analysis), resolution (over 2.5 between proteins), peak efficiency (theoretical plate numbers between 8 000 and 50 000), and a sample treatment without filtration steps that together with the analysis takes 2 h. The composition of protein from milk of each of 234 cows was determined using the current method, and the results were similar to reference values for milk proteins.