Eglin C is a polypeptide of 70 amino acids. Eglin C is a member of the family of serine proteinase inhibitors that have been shown to have therapeutic value. The use of peptides as therapeutic agents or as delivery agents for drugs and drug-peptide conjugates is becoming more common. Thus the quantitative measurement of the peptide from biological fluids is important to obtain pharmacokinetic data at low levels of detection. However, unlike small molecules, traditional liquid-liquid or precipitation methods often lead to poor recovery and precision for the extraction and HPLC/MS/MS analysis of peptides from plasma. Therefore, to obtain quality data, significant method development was warranted to develop a successful extraction technique used in combination with HPLC/MS/MS.