The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are time-consuming and low throughput. Thus, in high-throughput clinical contexts, there is a need for a technique that can assess cell malignancy rapidly. In this study, we assess the N:C ratio of four different malignant cell lines (OCI-AML-5-blood cancer, CAKI-2-kidney cancer, HT-29-colon cancer, SK-BR-3-breast cancer) and a non-malignant cell line (MCF-10A -breast epithelium) using an imaging flow cytometer (IFC). Cells were stained with the DRAQ-5 nuclear dye to stain the cell nucleus. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, 3159 SK-BR-3 cells, and 1109 MCF-10A cells. The N:C ratio was calculated as the ratio of the nucleus diameter to the total cell diameter. The average cell and nucleus diameters from IFC were 12.3 ± 1.2 μm and 9.0 ± 1.1 μm for OCI-AML5 cells, 24.5 ± 2.6 μm and 15.6 ± 2.1 μm for CAKI-2 cells, 16.2 ± 1.8 μm and 11.2 ± 1.3 μm for HT-29 cells, 18.0 ± 3.7 μm and 12.5 ± 2.1 μm for SK-BR-3 cells, and 19.4 ± 2.2 μm and 10.1 ± 1.8 μm for MCF-10A cells. Here we show a general N:C ratio of ~0.6-0.7 across varying malignant cell lines and a N:C ratio of ~0.5 for a non-malignant cell line. This study demonstrates the use of IFC to assess the N:C ratio of cancerous and non-cancerous cells, and the promise of its use in clinically relevant high-throughput detection scenarios to supplement current workflows used for cancer cell grading.