Accurate quantitation of microRNA (miRNA) in tissue samples is required for validation and clinical use of miRNA-based disease biomarkers. Since sample processing, such as RNA extraction, introduces undesirable biases, it is advantageous to measure miRNA in a crude cell lysate. Here, we report on accurate miRNA quantitation in crude cell lysate by a CE-based hybridization assay termed direct quantitative analysis of multiple miRNAs (DQAMmiR). Accuracy and precision of miRNA quantitation were determined for miRNA samples in a crude cell lysate, RNA extract from the lysate, and a pure buffer. The results showed that the measurements were matrix-independent with inaccuracies of below 13% from true values and relative standard deviations of below 11% from the mean values in a miRNA concentration range of 2 orders of magnitude. We compared DQAMmiR-derived results with those obtained by a benchmark miRNA-quantitation method-quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR-based measurements revealed multifold inaccuracies and relative standard deviations of up to 70% in crude cell lysate. Robustness of DQAMmiR to changes in sample matrix makes it a perfect candidate for validation and clinical use of miRNA-based disease biomarkers.