Objective The advantages and disadvantages of 12 and 24 h intervals of observation to evaluate the degree of estrous cycle disturbance were compared, and the significance of interphase sampling to evaluate a Tripterygium wilfordii-induced premature ovarian failure model in rat was explored. Methods Sixty female SD rats with a regular estrous cycle were randomly divided into blank groups 1 and 2, and Tripterygium wilfordii groups 1 and 2 (n= 15). The blank groups were gavaged with normal saline, and the TG groups were gavaged with a Tripterygium wilfordii polyside tablet (TG) suspension for 15 days. Blank group 1 and TG group 1 were observed for vaginal exfoliation cytology at 8:00 every day, and blank group 2 and TG group 2 were observed for vaginal exfoliation cytology at an interval of 12 h. Rats were weighed weekly. Rats with a regular estrous cycle were sacrificed on days 15 ~ 17 after treatment. Rats with an irregular estrous cycle were sacrificed on day 15 after treatment. Serum sex hormones were measured, ovarian and uterine indexes were calculated, and pathological changes of the ovary were observed. Results Compared with the black group, the degree of estrous cycle disorder in the TG group was increased. The number of normal estrous cycles was significantly decreased, the estrous period was significantly shortened, and the diestrus period was significantly prolonged when observed at an interval of 24 h. The difference between the two groups was better observed at 8:00 than at 20:00, and there was a significant difference in the level of estrous cycle disturbance (I and IV) between the two groups when observed at an interval of 12 h. Rats sacrificed in diestrus showed significantly decreased serum INHB and E2 in the TG group, while FSH was significantly increased. Rats sacrificed in any subcycle stage showed no significant differences between the groups. Because the serum AMH level fluctuated independently of the estrous cycle, the serum AMH level of rats in the TG group sacrificed in diestrus or any subcycle stage was significantly decreased. Conclusions Tripterygium wilfordii causes significant ovarian function decline, follicle development disorder, and ovarian reserve decline in rats. Cytological analysis of vaginal exudation at an interval of 12 h facilitates capturing cytological changes of the subcycle stage and reflects real changes of the estrous cycle. Disturbance of the estrous cycle in the model group observed at an interval of 12 h was better reflected than that observed at an interval of 24 h, but its feasibility needs to be further explored. Rats were sacrificed in diestrus, which reflected the basic sex hormone level and was beneficial to evaluate the success rate of model establishment of premature ovarian failure induced by Tripterygium wilfordii. Comparison of the ovarian pathological changes between the two groups in the subcycle stage objective ly and faithfully reflected the adverse effects of Tripterygium wilfordii on follicle development and luteal function.