The oviduct provides the optimal microenvironment for early embryo development. The composition of oviductal fluid has been studied for decades to elucidate embryo-maternal signaling responsible for successful reproduction, to optimize media for in vitro embryo production in assisted reproductive technologies and to understand the mechanisms underlying developmental programming. However, accessing the bovine oviductal fluid in vivo for analysis is still challenging and therefore the oviductal fluid is usually collected post mortem. The aim of the presented work was to utilize transvaginal endoscopy to gain minimal invasive access to the bovine oviduct for sample collection. We developed two novel methods to collect oviductal fluid protein in vivo. In the first experiment protein binding beads were transferred into the oviduct, oviductal fluid proteins were bound in situ and the beads were recollected by flushing. Proteins were eluted from the beads. In the second experiment oviductal fluid was collected by flushing the oviduct by repeated injection and aspiration of flushing solution. Protein concentration was determined by Bradford assay and 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 oviductal fluid proteome published. This new minimal invasive approach to access the bovine oviductal fluid facilitates future innovative experimental designs to study early embryo development.In a preliminary study oviductal fluid was collected post mortem to implement an in vitro culture system with oviductal fluid as the only protein source. The replacement of serum by oviductal fluid resulted in lower blastocyst rates, but higher post thaw embryo survival rates, indicating better blastocyst quality. In future studies oviductal collected in vivo could be used in in vitro culture to optimize embryo quality.