Phosphoprotein driven cellular signaling events represent most of the new molecular targets for cancer treatment. Application of reverse-phase protein microarray technology for the study of ongoing signaling activity within breast tumor specimens holds great potential for elucidating and profiling signaling activity in real-time for patient-tailored therapy. Analysis of laser capture microdissection primary human breast tumors and metastatic lesions reveals pathway specific profiles and a new way to classify cancer based on functional signaling portraits. Moreover, the data demonstrate the requirement of laser capture microdissection for analysis and reveal the metastasis-specific changes that occur within a new microenvironment. Analysis of biopsy material from clinical trials for targeted therapeutics demonstrates the feasibility and utility of comprehensive signal pathway activation profiling for molecular analysis.