Selected reaction monitoring (SRM) is a highly selective and sensitive mass spectrometric methodology for precise and accurate quantification of low-abundant proteins in complex mixtures and for characterization of modified peptides, and constitutes the method of choice in targeted proteomics. Owing to its outstanding features, SRM arises as an alternative to antibody-based assays for discovery and validation of clinically relevant biomarkers, a topic that is tackled in this article. Several of the obstacles encountered in SRM experiments, mainly those derived from shared physicochemical properties of peptides (e.g., mass, charge and chromatographic retention time), can compromise selectivity and quantitation. We illustrate how to circumvent these limitations on the basis of using time-scheduled chromatographic approaches and choosing appropriate spectrometric conditions, including the careful selection of the precursor and diagnostic ions.