Paper incorporated with ZnO nanostructures (called ZnO paper) is a flexible substrate material with applications in many areas including UV detectors and gas sensors. In this work, we tested the cytotoxicity of ZnO paper against cancer cells. The ZnO paper was placed in culture media of different cancer cell lines including A549, H1299, and WI38 with and without the targeted anti-lung cancer drugs Iressa and Staurosporine, and cell viability was determined. We found that ZnO nanoparticles were cytotoxic to all the cell lines examined, and cell viability was further reduced by increasing the deposition time of these nanoparticles. In addition, cytotoxicity was enhanced when both the ZnO paper and the anticancer drugs were present in the culture media. Our experimental results indicate that ZnO paper can be used for cytotoxicity test and the development of future novel anticancer drugs.