The Agilent 2100 Bioanalyzer can characterize in vitro RNA transcripts for their integrity, purity, concentration, and size. The results are comparable to denatured agarose electrophoresis with ethidium bromide staining and UV specirophotometry combined. In this report, we describe our strategy for validating this method following the International Conference on Harmonization guidelines. The assay has a linear range of quantitation between 500 and 25 ng/mu L. Quantitation accuracy is within +/- 20% of measurements produced from spectrophotometry and sizing accuracy is within +/- 7% based on theoretical sizes. Concentration and sizing measurements within a single assay produce RSDs that are <10 and <2%, respectively, indicating good precision. The method also maintains a tolerable precision when altering operator, day, and reagent kit lot. The RSD for quantitation is <= 25 and <2% for sizing. The LOQ and LOD are 15.4 and 5.4 ng/mu L based on experimentation, producing values similar to those specified by the manufacturer. The Bioanalyzer can differentiate between the RNA transcript and DNA contamination, and protein contamination quenches the RNA transcript signal. The effect of the ionic strength of the buffer on RNA analysis is also examined. Limitations of this method and future applications are discussed.