The oviduct provides the optimal micro milieu for early embryo development. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. In the study presented here we introduce a novel approach to gain minimal invasive access to the bovine oviductal fluid proteome in vivo by transvaginal endoscopy at different stages of the estrous cycle. The first experiment aimed at transferring C4 derivatised magnetic beads to bind the oviductal fluid proteome in situ. Protein carrying beads were recovered by flushing the oviduct and proteins were eluted. In the second experiment a flushing solution was injected into and aspirated from the oviduct repeatedly. The flushing solution was centrifuged to separate the fluid from the cellular debris. Proteins were identified by nano-LC-MS/MS. Two different stages of the estrous cycle (Day 1 and Day 3) were analyzed in samples from 30 heifers. Both methods were applied successfully and in total, more than 3000 proteins were identified, so far representing the most comprehensive OF proteome published. This new minimal invasive approach to access the bovine oviductal fluid proteome facilitates future innovative experimental designs to study the role of the oviductal micro environment during early embryo development.