Updated project metadata. SWATH-MS enables accurate and reproducible label-free quantification of proteomes including in extensively studied model organisms such as the mouse. Here we present a comprehensive mouse reference spectral library (MouseRefSWATH) that allows quantification of up to 10,597 proteins (62.2% of mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high protein sequence similarity between the mouse and human, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply XenoSWATH to characterise a xenograft model of breast ductal carcinoma in-situ and uncover complex regulation of cell migration pathways that is not restricted to the tumour cells but also operates in the mouse stromal upon progression to invasive disease. MouseRefSWATH and XenoSWATH opens new possibilities for in-depth proteomic assessment to address wide-ranging biological questions involving this important model organism.