We here present and demonstrate a novel technique based on isotachophoresis (ITP) for the quantification of global microRNA (miRNA) abundance in total RNA. We leverage the selectivity of ITP to concentrate miRNA and exclude longer RNA molecules from the focused zone. We designed a novel ITP strategy where we initially establish three contiguous zones of sieving polymer, electrolyte, and denaturant concentrations. This allows for successive preconcentration, selection, and detection of miRNA. We optimized chemistry in each zone for high sensitivity and exquisite selectivity for miRNA. This technique allows for the measurement of the total miRNA content in a sample and its comparison between different cell types and tissues. We demonstrated and validated the efficacy of this technique by comparing global miRNA abundance in subconfluent and confluent cell cultures.