Mass spectrometry based proteomics approaches are routinely used to discover candidate biomarkers. These studies often use small number of samples to discover candidate proteins that are later validated on a large cohort of samples. Targeted proteomics has emerged as a powerful method for quantification of multiple proteins in complex biological matrix. Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) are two main methods of choice for quantifying and validating proteins across hundreds to thousands of samples. Over the years, many software tools have become available that enable the users to carry out the analysis. In this chapter, we describe selection of proteotypic peptides, sample preparation, generating a response curve, data acquisition and analysis of PRM data using Skyline software for targeted proteomics to quantify candidate markers in urine.