Phosphorylation of proteins is an important cellular regulatory process. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of phosphorylated species. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests is the method-of-choice for characterization of phosphorylated proteins. However, determination of phosphopeptides by MS represents a major challenge, especially in the presence of unmodified peptides. Due to lower ionization efficiency of phosphopeptides, as well as the fact that the stoichiometry of phosphorylation is often present at low relative abundance, efficient enrichment of the phosphorylated peptides prior to MS analysis is therefore of high demand. In addition, successful identification of peptides with different phosphorylation grades still remains challenging. This work presents a new strategy for enrichment and subsequent selective elution of multi-, mono- and nonphosphorylated peptides based on their difference in pI by using pH gradient elution in presence of different concentration of acetonitrile prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis (MALDI-MS). The developed protocol was successfully applied for α-casein tryptic digest and bovine serum albumin digest spiked with 9 synthetic phosphopeptides. Further selectivity for phosphopeptides was demonstrated by fractionation of peptides from a milk digest.