Phosphorylation represents one of the most pervasive and meaningful post-translational modifications of proteins. The analysis of phosphorylation site occupancy is of great importance for elucidating protein function and disease mechanisms. Nevertheless, the direct mass spectrometric analysis of modification occupancy remains a significant challenge. In this study, a two-step derivatization method was employed in conjunction with liquid chromatography-tandem mass spectrometry for the examination of the phosphorylation site occupancy of peptides. The derivatization resulted in the specific labeling of phosphorylation sites and served to reduce the discrepancy in charge properties between phosphorylated and non-phosphorylated peptides, which is advantageous for their simultaneous detection. The occupancies of multiple sites from bovine casein and goldfish tissues were successfully estimated, confirming the usefulness and reproducibility of the method in the analysis of complex biological samples. Furthermore, the application of this method allowed for the observation of peptides with low occupancy, as well as peptides with markedly different occupancy levels between fish tissues. These results suggest that this methodology may be employed as a specialized analytical technique for site occupancy studies, thereby facilitating the investigation of the regulatory mechanisms that govern protein phosphorylation in biological processes.