The development of suitable analytical tools is required to study the stability, manufacturability and bio-performance of peptide conjugate therapeutics. Mass spectrometric analysis provides essential knowledge for understanding and building the critical quality attributes and regulatory specifications of peptides. The purpose of this chapter is to provide an overview/update of mass spectrometric techniques (e.g. ion stripping and isotopic labelling) that are employed for the analytical characterization and quantitation of peptides and their conjugates such as PEGylated peptides and antibody–drug conjugates. Advances in high-resolution mass spectrometry (HRMS) have considerably increased the quality of analytical data on antibody–peptide drug conjugates [e.g. average drug-to-antibody ratio (DAR), drug loading distribution, unconjugated drug and impurity characterization via peptide mapping] that can be used for product and process development, lot release and stability testing. Similarly, the quality of PEGylated peptides can be evaluated via qualitative and quantitative mass spectrometric analysis to ensure that the PEGylated therapeutics have the desired properties such as molecular mass and polydispersity. In addition, relative or absolute mass spectrometric-based quantitation of peptides/peptide conjugates is very useful for assessing stability, label claim and batch-to-batch variability. Different strategies exploiting stable isotope labelling and isotope dilution are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS) has recently emerged as a complementary technique to the previous methods allowing absolute protein quantification using suitable elemental-based tags.