A variety of RNA analysis technologies are available for the detection of RNA transcripts from bulk cell populations. However, the techniques for RNA detection from individual cells have been limited. Here we adapt a novel in situ signal amplification method (the RNAScope® detection platform) for the analysis of intracellular RNAs in individual cells by flow cytometry. Using novel target-specific probes that were designed to suppress background signals, we demonstrate the specific detection of HIV gag RNAs in HIV-infected cellular samples, in addition to bcr and abl mRNAs in the K562 cell line. This method was capable of distinguishing cells expressing low abundance RNA transcripts and correlated well with quantitative imaging analysis. Furthermore, multiple distinct RNA targets were simultaneously detected with a high specificity without interference. Overall, the sensitivity and specificity of this method will be useful for the analysis of functionally important RNA species from individual cells, even at very low copy numbers.