The endometrial fluid is a noninvasive sample which contains numerous secreted proteins representative of endometrial function and reflects the state of the endometrium. In this study, we describe, for the first time, a comprehensive catalogue of proteins of the endometrial fluid during the secretory phase of the menstrual cycle. To achieve this objective, three different but complementary strategies were used: First, in-solution digestion followed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS); second, protein separation by denaturing one-dimensional electrophoresis (SDS-PAGE) followed by HPLC-MS/MS analysis. Finally, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by MALDI-TOF/TOF analysis. The combination of the three strategies led to the successful identification of 803 different proteins in the International Protein Index (IPI) human database (v3.48). An extensive description of the endometrial fluid proteome will help provide the basis for a better understanding of a number of diseases and processes, including endometriosis, endometrial cancer and embryo implantation. We believe that the thorough catalogue of proteins presented here can serve as a valuable reference for the study of embryo implantation and for future biomarker discovery involved in pathologic alterations of endometrial function.