In vitro embryo production systems in buffaloes have great productive perspectives and opportunities for improvement. Among these, comparative studies with species with more significant advances in reproductive biotechnology have been developed. Accordingly, this work aimed to identify the differences in the electrophoretic profiles of proteins in the follicular fluid (FF) of cows and buffaloes and their possible association with follicle size. FF was obtained at the central abattoir in Medellín (Antioquia), Colombia, from small (<7 mm) and large (>7 mm) follicles from the ovaries of 25 cows (Bos indicus) and 20 buffaloes (Bubalus bubalis). The total protein content of the FF was quantified and subsequently depleted of albumin and immunoglobulins. Samples were subjected to denaturing electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) to determine the electrophoretic profiles using a photodocumenter. The values obtained for the relative amount of each band were compared between species and follicle sizes using the Mann-Whitney test. The results showed no significant differences in total protein concentration between the different follicle sizes and species. Further, 72.6% of the FF proteins are immunoglobulins and albumin. The profiles of small follicles (<7mm) in cows presented 19 bands and 11 in buffaloes. The molecular weight range of the bands detected was between 5 and 250 kDa. Quantitative differences of the proteins in the follicular fluids evaluated were identified. The information obtained may contribute to elucidating the physiological differences between large and small follicles but does not explain the differences between species.