This study provides a comprehensive investigation of TrEnDi for phosphopeptide analysis using mass spectrometry (MS). The goal is to broaden the scope of TrEnDi in phosphopeptide research, emphasizing its versatility and efficacy in peptide analysis. The initial phase involved applying TrEnDi to synthetic peptides, FLEEpXK (X= S, T or Y) and STEALMYCAR, to assess its impact on phosphate and sulfur-containing residues. Through iterative optimization, involving the synthesis of different phosphopeptides and LC-MS parameters, enhanced sensitivity and peptide retention were achieved. The successful application of TrEnDi to synthetic peptides prompted its evaluation on myoglobin and stathmin. TrEnDi-modified peptides demonstrated improved ionization efficiency, retention times, and sensitivity, confirming the successful derivatization. The study highlights future avenues, including exploring N-terminal phosphorylated residues, extending TrEnDi to diverse phosphoproteins, establishing a dedicated database, and integrating it with other methodologies. This research highlights TrEnDi's potential as a valuable tool for comprehensive phosphoproteomic analysis in conjunction with MS.