Inherent interindividual and intraindividual variation in the length of the menstrual cycle limits the accuracy of predicting days of peak fertility. To improve detection of days of peak fertility, a more detailed understanding of longitudinal changes in cervicovaginal fluid (CVF) biomarkers during the normal menstrual cycle is needed. The aim of this study, therefore, was to characterize longitudinal changes in CVF proteins during the menstrual cycle using a quantitative, data-independent acquisition mass spectrometry approach. Six serial samples were collected from women (n = 10) during the menstrual cycle. Samples were obtained at two time points for each phase of the cycle: early and late preovulatory, ovulatory, and postovulatory. Information-dependent acquisition (IDA) of mass spectra from all individual CVF samples was initially performed and identified 278 total proteins. Samples were then pooled by time of collection (n = 6 pools) and analyzed using IDA and information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]). The IDA library generated contained 176 statistically significant protein identifications (P < 0.000158). The variation in the relative abundance of CVF proteins across the menstrual cycle was established by comparison with the SWATH profile against the IDA library. Using time-series, pooled samples obtained from 10 women, quantitative data were obtained by SWATH analysis for 43 CVF proteins. Of these proteins, 28 displayed significant variation in relative abundance during the menstrual cycle (assessed by ANOVA). Statistical significant changes in the relative expression of CVF proteins during preovulatory, ovulatory, and postovulatory phases of menstrual cycle were identified. The data obtained may be of utility not only in elucidating underlying physiological mechanisms but also as clinically useful biomarkers of fertility status.