Development of novel approaches for quantitative analysis of gene expression in intact tumor cells should provide new means for cancer detection and for studying the response of cancer cells to biological and therapeutic reagents. We developed procedures for detecting the levels of expression of multiple genes in fixed as well as viable cells using molecular beacon imaging technology. We found that simultaneous delivery of molecular beacons targeting survivin and cyclin D1 mRNAs produced strong fluorescence in breast cancer but not in normal breast cells. Importantly, fluorescence intensity correlated well with the level of gene expression in the cells detected by real-time reverse transcription-PCR or Western blot analysis. We further show that molecular beacons can detect changes of survivin gene expression in viable cancer cells following epidermal growth factor stimulation, docetaxel treatment, and overexpression of p53 gene. Thus, molecular beacon imaging is a simple and specific method for detecting gene expression in cancer cells. It has great potential for cancer detection and drug development.