RNA-Seq is an approach to transcriptome profiling that uses deep-sequencing technologies to detect and accurately quantify RNA molecules originating from a genome at a given moment in time. In recent years, the advent of RNA-Seq has facilitated genome-wide expression profiling, including the identification of novel and rare transcripts like noncoding RNAs and novel alternative splicing isoforms. Here, we describe the analytical steps required for the identification and characterization of noncoding RNAs starting from RNA-Seq raw samples, with a particular emphasis on long noncoding RNAs (lncRNAs).