This study evaluated the effects of serum (egpS) and follicular fluid (egpFF) from estrus guinea pigs as in vitro maturation (IVM) supplements for natural cycle guinea pig oocytes. Two experiments were conducted: the first assessed three IVM media, a commercial medium (CMOM) and two homemade media (HMOM-P and HMOM-S), for oocyte in vitro maturation using 615 bovine oocytes, while the second evaluated the incorporation of 5%, 10%, and 20% egpFF or egpS into IVM media for 1744 guinea pig oocytes. Initially, we optimized the IVM base medium using bovine oocytes to determine the most suitable culture conditions. The results obtained from these experiments served as a critical foundation for subsequent supplementation trials conducted with guinea pig oocytes. The oocytes were cultured in 70 μL drops under controlled atmospheric conditions, and maturation rates were assessed based on cumulus cell expansion and nuclear progression. The HMOM-S medium significantly enhanced cumulus cell expansion (72.5 ± 2.88%) compared with the CMOM medium (57.7 ± 5.33%