A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. While gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix-assisted laser desorption/ionization) mass spectrometry directly on uterine sections, we provide here molecular composition, relative abundance and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region and stage specific manner with the progression of implantation. This application is reliable since patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cPLA₂ null females that show implantation defects.