Abstract Introduction Female sexual behavior animal translation data do not translate to acute results and neither current synchronization invasive models (ovariectomy+Estrogen “E2”+Progesterone “P4”) guarantee hormonal physiological values. Objective Therefore, our study investigated the effect of non-invasive oestrus synchronization on sexual behavior. Methods Methods: Twelve week old Sprague Dawley intact rats were synchronized and randomly divided into three groups each of n=8-10. Each group received a dose of prostaglandin F2α “PGF2α” (75, 125 and 250 μg) at days 0 and 3, and 1 mg of progesterone at day four. Vaginal cytology was performed to determine the point of the cycle and the animals underwent an incentive test. To investigate the sexual behavior, the synchronized female rats were paired with trained male rats, and behavior recorded. The videos were quantified by trained operators. Sexual behavior was quantified as paracopulatory (hops, darts, ear wiggling, sniffing from male and from female) or copulatory (male mounting female, thrusting from male, intromission, ejaculation, female for sniffing position, lordosis, orgasm, female mounting on male, female rotation in her axis and female licking itself), E2 and Follicle Stimulating hormone (FSH) in plasma and E2 in vaginal fluid were quantified by immune assay. Results Based on the vaginal cytology, the proportion of animals in proestrus (p), estrus (e) or metestrus (m) was calculated with values of 0.5 (p), 0.25 (e) and 0.25 (m) for the group which received 0.75 μg PGF2α; 0.4 (p), and 0.6 (e) for the group which received 0.125 μg PGF2α; 0.2 (p), 0.5 (e), and 0.3 (m) for the group which received 250 μg PGF2α. The numbers of hops was higher in the proestrus compared to the metestrus, and of mounts higher in the estrus compared to the metestrus phase. The sexual encounter profile and E2 levels were unchanged with the different PGF2α doses, but in the high-dose group no hops were observed. The levels of sexual behavior was comparable to previous studies of hormone substituted ovariectomized rats, but E2 and FSH plasma levels were in a physiological range. Conclusions In summary, the non-invasive synchronization protocol could substitute the state-of-the-art used ovariectomized+hormone (E2) model. Our findings suggest the largest part of rats were synchronized in the oestrus phase with 125 μg PGF2α. but the 250 μg had also a positive profile. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Initiator Pharma A/S.