Health-promoting peptides are bioactive molecules derived from various natural protein sources, which may include dairy, meat, plant, and marine proteins. These peptides exhibit significant physiological health benefits such as antioxidant, antihypertensive, antimicrobial, and anti-inflammatory activities, which make them valuable for application in nutraceutical, pharmaceutical, and functional foods. The extraction and quantification of these peptides are critical steps for utilizing them for therapeutic use. This chapter provides an overview of the methods used for the extraction and quantification of health-promoting peptides. In enzymatic hydrolysis the utilization of proteolytic enzymes such as trypsin, pepsin, and alcalase is there, this is a most widely used method because of its specificity and efficiency to yield bioactive fragments. The quantification strategies emphasizing spectrophotometric assays, mass spectrometry (MS), etc. are the analytical tools which are essential for characterizing peptide sequences, molecular weights and biological acitivities. In this chapter, the recent advancements and challenges in peptide extraction and quantification have been highlighted.