RNA interference (RNAi) is a sequence-specific gene regulatory mechanism in which the specificity is determined by small RNAs. Three major classes of endogenous small RNAs, namely microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs/gsRNAs), have been characterized in vertebrates. The miRNAs are mainly involved in development and differentiation and alter gene expression through translational repression or mRNA cleavage. The siRNAs, in contrast, mainly defend against molecular parasites including viruses, transposons, and transgenes. We reported on the expression profile of miRNAs during Xenopus development using a combination of cloning and Northern blot analysis of stage-specific small RNAs. The expression of most miRNAs appeared to be regulated, and some were only expressed at specific stages of development. We also reported on small RNAs specifically expressed during gametogenesis in the mouse. The study revealed the existence of retrotransposon-derived siRNAs in oocytes and a novel class of small RNA (piRNAs/gsRNAs) in testes. In this chapter, we describe methods of low molecular weight RNA preparation, small RNA cloning, annotation of small RNAs, and analysis of expression during development. © 2008 Humana Press.