Discovery of non-coding RNAs (ncRNAs) has led to a drastic change in the understanding of the central dogma of molecular biology. These molecules play a complex regulativory role in genome organization and gene expression. Particularly, the small ncRNAs, are responsible for controlling fundamental processes such as development, physiology, and various defence responses in plants and animals. Multiple in silico approaches have been designed to predict and characterize different types of ncRNAs. To validate them experimentally, traditional methods such as quantitative-real time PCR (qPCR) and northern blot are most commonly utilized; but these have been limited to some extent. Advanced methods such as RNA seq, in-situ hybridization, RLM-RACE, PARE, luciferase assays, and pulldown approaches have been of great importance. These methods are quite efficient in identifying the numerous transcripts, their interactions and functional complexes, which can give a detailed view of their roles in biological processes. Functional analysis of ncRNAs adds a new paradigm in the understanding of RNAi machinery to draw some useful solutions for the ongoing challenges in food production, medical sciences, and industrial applications. This chapter focuses on the methods employed for the characterization of known and novel ncRNAs.