Endometrial cancer is the most common gynaecological malignancy in high-income countries and its incidence is rising. Early detection, aided by highly sensitive and specific biomarkers, has the potential to improve outcomes as treatment can be provided when it is most likely to effect a cure. Sequential window acquisition of all theoretical mass spectra (SWATH-MS), an accurate and reproducible platform for analysing biological samples, offers a technological advance for biomarker discovery due to its reproducibility, sensitivity and potential for data re-interrogation. SWATH-MS requires a spectral library in order to identify and quantify peptides from multiplexed mass spectrometry data. Here we present a bespoke spectral library of 154,206 transitions identifying 19,394 peptides and 2,425 proteins in the cervico-vaginal fluid of postmenopausal women with, or at risk of, endometrial cancer. We have combined these data with a library of over 6,000 proteins generated based on mass spectrometric analysis of two endometrial cancer cell lines. This unique resource enables the study of protein biomarkers for endometrial cancer detection in cervico-vaginal fluid.