Toxicity of engineered nanomaterials (ENMs), such as metal oxides, has been of concern among environmental and health scientists. For ecotoxicity studies of ENMs, it is important to assess nanoparticle uptake and correlate it with the cellular response. However, due to nonavailability of adequate methods for assessing cellular uptake of ENMs, there is a lack of information in this important area. In the present study, a method has been developed using flow cytometry, which allows for rapid detection of ENM internalization in live bacteria under different experimental conditions for several generations. Our data demonstrate significant internalization of Zinc oxide (ZnO) and Titanium (IV) oxide (TiO(2) ) nanoparticles (NPs) in Escherichia coli in a dose-dependent manner. ZnO NPs treatment exhibited a significant increase in the intensity of side scatter (SSC) with liver-S9 fraction (76, 94, and 181% increase) rather than without S9 (10.5, 24.5, and 125.9% increase) at 10, 40, and 80 μg/ml concentrations, respectively. This was due to the protein coating of NPs by the S9 fraction. A similar response was also observed on exposure to TiO(2) NPs (139 and 203% with S9 and 128 and 198% without S9). In a multigeneration study, this new method was able to detect the presence of ENMs in E. coli up to four generations. Our data demonstrate that this method can be used for assessing the uptake of ENMs in bacteria and provides a handle to toxicologists for ecotoxicity studies of economically important ENMs to ensure safer products in the market.