We have developed a simple method for the quantification of milk fat in foods using 1,2-dipalmitoyl-3-butyroyl-glycerol (PPBu) as an indicator of milk fat content by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry. The separation of the triacylglycerol positional isomer, 1,3-dipalmitoyl-2-butyroyl-glycerol (PBuP) and PPBu, was achieved using an octacocyl silylation (C28) column, and multiple reaction monitoring was employed. The milk fat contents in butter, butter-blended margarine, and butter cookies were quantified using two different sample preparation methods. In the first method (Method A), the lipid in the food was extracted with organic solvents and used for the preparation of a sample solution. In the other method (Method B), the sample solution was prepared by dissolving the food in organic solvents; the PPBu content in the fat and oil was corrected by the lipid content in the food obtained by the rapid NMR method. The calibration curve of standard PPBu was a first-order equation over the range of 1-250 μg/mL. The recovery rates of PPBu spiked into butter evaluated by Methods A and B were 99.9-105.0% and 106.5-110.1%, respectively. PBuP was not detected in milk fat. The milk fat contents in blends of butter and margarine determined by the method developed in this study were equivalent to the contents calculated with the butyric acid (Bu) method using GC-FID. The milk fat contents in butter-blended margarine analyzed by Methods A and B were almost the same. The PPBu content in blends of butter and margarine was correlated with the butter content. Thus, we succeeded in developing an efficient method for the rapid quantification of milk fat content and the detection of milk fat adulteration.