The utilization of peptides as therapeutic agents has evolved over the past two decades. The earliest drugs revolved around native structures such as glucagon and insulin, which were limited by their poor ADME properties including instability, and rapid proteolytic cleavage. Developing longer acting peptides was initially attempted by incorporation of unnatural amino acids to block metabolism. Significant advances have resulted in longer acting peptides employing modifications to increase protein binding or decrease renal clearance. These structural changes including conjugation of peptides with PEG, lipids, EXTEN, and FC-fusion, create unique analytical challenges. Analytical methods for native peptides were comparable to small molecule assays by mass spectrometry. The development of higher molecular wt. peptides and conjugate technology made intact analysis impractical. Digestion of peptides followed by detection of tryptic fragments became the main stream for analysis. Issues with this technique included monitoring peptide fragments not representative of active or intact peptide. With the advent of high resolution mass spectrometry in 2005, intact peptide analysis became possible, eliminating these uncertainties. In addition to instrumental challenges, traditional extraction techniques for high protein binding peptides such as protein precipitation, SPE and liquid/liquid suffer from low extraction efficacy. In most cases, immunoprecipitation is the only option to achieve detection limits of 1 ng/ml or lower. Recent developments in extracting peptides with high protein binding have allowed improved detection levels without the need for expensive immunoprecipitation reagents. In summary, this talk will outline the evolution of therapeutic peptide structure as it relates to analytical challenges.