This protocol describes a method that guides researchers in designing assays for any miRNA of interest, andalso allows researchers to easily recover and quantify miRNAs without reliance on expensive proprietary kits. Using our novel assay design, we have previously shown that it is possible to effectively discriminate mature miRNAs from their precursors and almost identical homologs in multiplex. We here extend the approach to include very recently sequenced miRNAs for which no commercial assays are yet available yet. We further show that efficient lysis, recovery and detection of miRNAs are achievable with common reagents and basic enzymatic kits.