RNA interference (RNAi) has become a powerful biological tool over the past decade. In this study, a tetracycline-inducible small hairpin RNA (shRNA) vector system was designed for silencing cyan fluorescent protein (CFP) expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% +/- 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene-silencing effects using RNAi in single cells and cell populations.