Research in humans has long recognized a modulating role for sex hormones on fear learning. There is still not quite a consensus on what specific effects different hormones have, or what experimental paradigm properly reflects key processes of fear-related learning in near-ecological situations. Our aim with this work was to precisely tackle the influence of sex hormones and the menstrual cycle in fear memory, combining for the first time methods such as vaginal cytology analysis, liquid chromatography-mass spectrometry (LC-MS), fear-potentiated startle (FPS) and skin conductance response (SCR). We developed a differential fear conditioning and delayed (24 hours after) fear extinction task, to take into account the memory consolidation window. Participants were allocated into 5 experimental groups considering sex and hormonal status: men, early follicular women, late follicular women, mid luteal women, and women taking oral contraceptives. Moreover, 17 hormones were quantified in saliva samples using LC-MS, including not only sex steroids but also glucocorticoids like cortisol and cortisone. We found better fear extinction patterns in late follicular women in comparison to early follicular women, as well as an impairment in fear acquisition shown by mid luteal women. These results were supported by analyses with quantified estradiol and progesterone concentrations. In summary, our study shows sex differences in fear memory, shining light on menstrual cycle and sex hormones impact and guiding future research while emphasizing the need for a precise multidimensional approach to the study of this topic.