Development of the capability to enable large-scale proteome studies, analogous to comprehensive gene expression analysis, will clearly have far-reaching impacts on protein biomarker investigations of human diseases such as cancer through interrogation of the archived fresh frozen and formalin-fixed and paraffin-embedded tissue collections. This review therefore focuses on the most recent advances in microdissection techniques and proteome platforms for procuring homogeneous subpopulations of tumor cells or structures and performing comprehensive analysis of protein profiles within tissue specimens, respectively. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated tissue proteome effort. The capabilities of CIEF-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. These proteome technological advances combined with recently developed tissue microdissection techniques provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.