Peptidomic analysis and methodological development of characterization of low molecular weight peptidome of human wound fluids. Wounds are dynamic environments with high proteolytic activity. Therefore, the study of peptides, being the degradational product of proteins, are of interest when diagnosing infection as well as understanding wound physiology. Three types of wound fluids are studied: acute (sterile), infected and non- infected (unsterile from facial skin graft surgery). We have provided a methodology for sample preparation and analysis, as well as the first quantification of wound fluid peptidomes using LC-MS/MS data, sorting algorithms and 3 rd party software. Furthermore, we visualize the differences between the abovementioned peptidomes and create a methodology for peptidomic analysis of LC-MS/MS data. We report several findings regarding the general characteristics, protease environment and P1-P1’/P4-P4’ prominence in the various fluids. Additionally, we find that there is an abundance of antimicrobial peptides derived from hemoglobin subunit beta in infected wounds. Furthermore, we crosscheck our data with existing literature, quantifying abundance of potential biomarkers.