The interest in graphene and its translation into commercial products have been expanding at high pace. In regard to previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and pulmonary system. The aim of present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions – s-GO (50 – 200 nm) and l-GO (5 – 15 µm) were produced and thoroughly characterized. Exploiting intrinsic fluorescence of GO and using confocal live-cell imaging, we visualized the behavior of the cells in response to the material in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated ROS levels, pro-inflammatory response and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, lateral dimension of GO played more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO induced cytotoxicity.