Graphene and other 2D materials have received increased attention in the biomedical field due to their unique properties and potential use as carriers for targeted drug delivery or in regenerative medicine. Before the exploitation of graphene-based materials in biomedicine becomes a reality, it is necessary to establish the full toxicological profile and better understand how the material interacts with cells and tissues. Because specific properties, such as flake size and surface chemistry, might determine whether graphene can achieve therapeutic efficacy without causing toxicity, it is important to develop highly accurate and reliable screening techniques to accurately assess the biocompatibility of different types of graphene-based materials. In this protocol, we describe a method to achieve accurate determination of the cytotoxic response following in vitro exposure to large graphene oxide (L-GO) sheets using annexin V/propidium iodide staining and the Imagestream® platform. The proposed protocol is especially suitable for the toxicity assessment of carbonaceous materials that form aggregates in cell culture media, which is a common occurrence. We describe how to best gate-out any interfering signals coming from the material by visual inspection and by using powerful software, thus performing the analysis of cellular death on a selected population of cells with higher accuracy and statistical relevance compared to conventional flow cytometry.