Selected reaction monitoring (SRM) is one of the most powerful techniques for the relative and absolute quantification of proteins from complex protein mixtures. In contrast to traditional protein quantification methods such as ELISAs or RIAs, the SRM method uses mass spectrometry for detection. Further benefits of SRM are as follows: (1) high specificity and sensitivity; (2) large linear dynamic range of at least three orders of magnitude; and (3) the possibility to quantify multiple proteins simultaneously in a single MS run from an individual sample. To perform SRM-based protein quantification reliably, a careful design of the assay is essential, and several pitfalls must be avoided. The aim of this chapter is to help SRM newcomers to establish SRM-based protein quantification assays and discuss an overview of typical work flows that are applied during SRM assay development.