Reversed-phase high-performance liquid chromatography (RP-HPLC) is a cornerstone technique for peptide analysis due to its exceptional resolving power. In this webinar, we will explore how to optimize RP-HPLC parameters for effective separation of peptides derived from protein digests and analysis of PTMs analysis. Key topics include the role of stationary phase chemistry in influencing selectivity, and how column physical characteristics affect separation performance. We will also examine how mobile phase modifiers—such as trifluoroacetic acid (TFA), formic acid, and pH—impact chromatographic outcomes and compatibility with mass spectrometry (MS). Attendees will gain practical insights into method development strategies that enhance both resolution and detection sensitivity.