Peptide therapeutics represents one of the fastest-growing sectors in the pharmaceutical drugs pipeline, with an increasing number of regulatory approvals every year. Their pharmacological diversity, biocompatibility, high degree of potency and selectivity make them an attractive choice in several therapeutic areas, such as diabetes, cancer, immune, metabolic, cardiovascular and infectious diseases. However, the development of peptides as drugs presents its own set of challenges, necessitating extensive property optimization aimed at improving their drug-like properties and stability in biological environments. The discovery and development of innovative peptide therapeutic platforms entail the employment of several biophysical techniques, which monitor the structural as well as the functional integrity of peptides. Small structural changes of the bioactive peptides in response to the presence of various excipients can have a major impact on their pharmaceutical prowess, necessitating the use of analytical techniques for efficient quality control during development. Here we present some widely used methods, such as circular dichroism, fluorescence spectroscopy and multi-dimensional homo and heteronuclear nuclear magnetic resonance spectroscopy that form an integral part of therapeutic peptides development. The application of combination biophysical platforms ensures the maintenance of the appropriate folded structure, which is a prerequisite for the safety and efficacy of peptide pharmaceuticals.