Unlike the situation in humans, no specific biomarker has been related to early pregnancy in dogs. Such a biomarker would be beneficial for animal welfare reasons, e.g. in cases when the bitch has been mismated, and would also be of value for the breeder, to enable better planning for an expected litter. In the present study, a shotgun proteomic analysis based on nano-liquid chromatography coupled to high resolution MS was performed to identify differentially expressed proteins during canine pregnancy, and thereafter, a targeted PRM method was developed and applied to absolutely quantify the concentrations of a selection of them.