Abstract Lab animals, such as Guinea pigs (Cavia porcellus), are crucial for scientific development, as they play an important role in the development and quality control chain of vaccines and drugs distributed by the Brazilian public health system. Investigating their biological and physiological parameters is fundamental to raise and keep these animals, so the handling of the facilities that hold them can be updated whenever new information comes up, with the well-being of the animals and alignment with the 3 Rs in mind. In the search for understanding reproductive aspects of Guinea pigs, the present study had the main goal of studying puberty by means of estrous cycle analysis in short-haired Guinea pigs. Guinea pigs have a vaginal occlusive membrane that covers the vaginal orifice. Its rupture takes place gradually and naturally, moments before labor and during estrus. The present study followed 42 females as for the presentation of the vaginal occlusive membrane. Once the membranes ruptured spontaneously, a swab was collected to study vaginal cytology. Membrane rupture was observed in 39 females; six females showed membrane rupture with less than 21 days of age (17 to 21 days). Twenty-three females were characterized as being in estrus due to cytology showing a prevalence of anucleated superficial cells. One of these females was younger than 21 days old. The opening of the vaginal occlusive membrane took place most frequently in intervals between 17 and 18 days, and the membrane remained open between one and three consecutive days. It was possible to follow three cycles of membrane opening on six females. The present study showed the need to adapt handling guidelines for C. porcellus kept in research animal facilities. The early age of puberty imposes the need of separate the female daughters from their fathers at 16 days old.