The rapidly growing list of small RNA species generated by next-generation sequencing technologies has accelerated the development of new bioinformatics tools for their detection. Small RNAs generated from tRNAs, transfer RNA-derived fragments (tRFs), represent a novel challenge in accurately identifying and distinguishing them from random degradation products of tRNAs. Here, we describe a bioinformatics approach to detect tRFs in next-generation sequencing libraries. We also present a biochemical purification protocol for enriching 5' tRFs and separating them from miRNAs. And finally, we suggest reliable methods for detecting and quantifying tRFs.