Harnessing the RNA interference (RNAi) process with chemically synthesized small interfering RNA (siRNA) is dependent on the development of efficient delivery vehicles that can help overcome the numerous barriers existing for siRNA delivery. However, quantifying the intracellular amount of siRNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular siRNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol for accurate and sensitive determination of the effective intracellular siRNA concentration upon transfection with different reagents. Specific guidelines for the customization of the protocol are provided and reported together with an example of its application for studying a specific siRNA delivery case. The outcome of the present study is a thoroughly discussed analytical protocol generally applicable to characterize carrier-mediated siRNA delivery processes.