Human ovulation is a biologically complex process that involves several biochemical factors, promoting follicular rupture and release of a fertilizable oocyte. Proteins which are present in follicular fluid at high concentrations during ovulation are likely to be active participants in the biochemical pathways of ovulation. The aim of the study was to identify, by use of modern proteomic technique, proteins of human follicular fluid which are differentially regulated during ovulation of the natural menstrual cycle.This prospective experimental study over three years included women planned for laparoscopic sterilization. During surgery, retrieval of the dominant follicle was performed, either at the preovulatory stage or during ovulation. Four women of preovulatory phase and four women of ovulatory phase met the predetermined criteria of hormone levels for respective phases, and samples of these were finally included out of 15 women operated. Follicular fluid was aspirated from the excised follicle and subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides. This enables simultaneous identification and quantification of proteins. The protein profiles of the follicular fluid of the preovulatory phase and the ovulatory phase were analyzed, and present proteins were identified.A total of 502 proteins were identified, several of which previously have not been identified in human follicular fluid. Out of the 115 proteins that were found in all samples, 20 proteins were at higher levels during ovulation. These were inflammatory-related proteins, coagulation factors, proteins in lipid metabolism, complement factors and antioxidants. Five proteins were present in lower levels during ovulation, with three being enzymes, and the other two proteins being proteins of lipid metabolism and iron transport.Twenty-five follicular-fluid proteins, with differential regulation during ovulation, were identified in human follicular fluid of the natural menstrual cycle. These proteins may carry essential roles in the ovulatory cascade.