The β-casein phosphopeptide 1-25 (βCPP) is involved in calcium binding, cellular transduction, and dental remineralization. The objective of this work was to improve upon the original protocol commonly used for isolation of this phosphopeptide from β-casein. This method exploits the isoelectric point of β-casein fragments to selectively precipitate βCPP. The highest βCPP extraction yield reported to date with this protocol is 14.4±0.5% of theoretically available βCPP. The present work optimizes 2 steps in this procedure, namely the length of trypsin digestion and incorporation of cold acetone precipitation, to increase the yield to 32.3±5.4%. Reverse-phase HPLC indicated high purity of the isolate, whereas mass spectrometry confirmed 2 forms of the phosphopeptide: fragments 1-25 (87%) and 2-25 (13%). The adaptation of the existing protocol represents a significant improvement in extraction yield and facilitates preparation of larger amounts of high-purity βCPP for subsequent analysis and use in functional foods and other applications.