To facilitate analysis of protein expression changes in in situ tumors and stroma, we took advantage of a mouse model that permits conditional activation of the Ser-Thr kinase ROCK within mammary tumor cells. In this study, we undertook MALDI-MSI analysis of tissue samples derived from our conditional ROCK mammary tumor model, to quantify in an unbiased manner, the proteomic changes occurring during the progression of mammary cancers in their specific spatial contexts.