Objective To find a simple staining method to determine the four phases of the estrous cycle, vaginal smear cells from female Sprague-Dawley (SD) rats. Methods Twenty female SD rats were randomly divided into two groups (10 each). The inclusion criteria were age 10 -13 weeks and weight 216 - 234 grams (mean: 225 ± 6 g). Rat vaginal smears were checked daily for 6 days. A wet cotton swab was inserted into the vaginal canal at 0. 5 -1. 0 cm deep,gently turned once or twice, and then placed on a clean glass slide with a saline solution. After air drying, the sample was stained. Rapid Gram and crystal violet staining was performed. The staining was detected under a microscope to determine the rat estrous cycle. Results The cell nuclei were stained deep purple, cytoplasm was lilac, and leukocytes were deep purple in crystal violet staining. The cell nuclei were stained amaranth, cytoplasm was rose red, and leukocytes were darkly red in rapid Gram staining. The estrous cycle in female rats averaged 4 -5 days. The estrous cycle is divided into different four phases: proestrus, estrus, metestrus, and diestrus. During proestrus, cells were large round, well-formed nucleated epithelial cells. During the estrus, cells were almost all large polygonal cornified squamous epithelial cells packed into clusters. During the metestrus, small and round leukocytes predominated, and nucleus-free keratinocytes were present.During the diestrus, there were three kinds of cells, mainly small leukocytes. Both crystal violet and rapid Gram staining could determine the four stages of the estrous cycle in female SD rats. The two staining method were simple, and the results were similar. However, the morphology of the crystal violet-stained cells was sometimes deformed slightly. The cell morphology of the rapid Gram staining method was complete, and the images were clear. Rapid Gram staining was timeefficient without fixing by absolute ethyl alcohol. Conclusions Vaginal smears with rapid Gram staining are a simple approach to easily and quickly determine the phases of the estrous cycle in female SD rats without changing their reproductive cycle. This method can be applied to large scale experiments.