Increasing lines of evidence indicate that small non-coding RNAs including miRNAs, piRNAs, rasiRNAs, 21U endo-siRNAs, and snoRNAs are involved in many critical biological processes. Functional studies of these small RNAs require a simple, sensitive, and reliable method for detecting and quantifying levels of small RNAs. Here, we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.