The search for novel and clinically relevant biomarkers still represents a major clinical challenge and mass-spectrometry-based technologies are essential tools to help in this process. In this application, we demonstrate how selected reaction monitoring (SRM) can be applied in a highly multiplexed way to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Such an assay can be used to analyze numerous samples for narrowing down a list of potential biomarkers to the most relevant candidates. The use of FFPE tissues is of high relevance in this context as large sample collections linked with valuable clinical information are available in hospitals around the world. Here we describe in detail how we proceeded to develop such an assay for 200 proteins in breast tumor FFPE tissues. We cover the selection of suitable peptides, which are different in FFPE compared to fresh frozen tissues and show how we deliberately biased our assay toward proteins with a high probability of being measurable in human clinical samples.