This review presents an overview and recent progress of strategies for detecting isomerism in peptides, with focus on d/l epimerization and the various isomers that the presence of an aspartic acid residue may yield in a protein or peptide. While mass spectrometry has become a majorly used method of choice within proteomics, isomerism is inherently difficult to analyze because it is a modification that does not yield any change in mass of the analyte. Here, several techniques used for analysis of peptide isomerism are discussed, including enzymatic assays, liquid chromatography, and capillary electrophoresis. Recent progress in method development using mass spectrometry is also discussed, including labeling strategies, fragmentation techniques, and ion‐mobility spectrometry.