In this chapter, we focus on issues related to the application of next-generation sequencing (NGS) strategies for the analysis of genes with pseudogenes in a clinical setting. Hereby, target enrichment and mapping strategies are crucial factors to avoid "contaminating" pseudogene sequences, which are characterized by higher mutation rates than their functional parental genes. For the target enrichment strategies, we describe advantages and disadvantages of PCR- and capture-based enrichment methodologies. For the mapping strategies, we discuss crucial parameters that need to be considered to accurately distinguish sequences of functional genes from pseudogenic sequences. Finally, we discuss some concrete examples of genes with known pseudogenes and associated with Mendelian disorders that were analyzed by NGS on various platforms and starting from different library preparations.