Small RNAs (sRNAs) are known to play an important regulatory role in a wide range of organisms and biological processes through expression regulation of a diverse array of genes. Several classes of sRNA including plant microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) carry an O-methyl modification at their 3´ termini, and this is suspected to complicate their accurate quantification. miRNA precursors have been detected in cell lines and other tissues, where their amount does not necessarily correlate with the amount of their mature miRNA due to different regulation processes controlling their biogenesis. Methods currently used to identify and quantify sRNAs face unique challenges due to their short length, as well as the high sequence similarities between them. The correct identification, discrimination and profiling of the different types of sRNAs, including their available isoforms, are crucial for understanding of the regulatory networks they are involved in.