Macromolecular transport between the nucleus and the cytoplasm is central to a myriad of eukaryotic cellular processes. This process depends on facilitated diffusion through nuclear pore complexes (NPCs) which selectively allow permeation of cargoes bound to nuclear transport receptors while blocking most other large cargoes. The nucleocytoplasmic transport machinery is subject to various physiological and pathological regulation. For example, several types of viruses, including SARS-CoV-2, manipulate the nucleocytoplasmic transport machinery to nullify the innate anti-viral defense of the host cells. However, there has been no quantitative assay that measures the nucleocytoplasmic transport kinetics in live cells in a high-throughput manner, limiting a deeper mechanistic understanding of such regulations and translational research. To overcome this obstacle, we developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. In our recent publication, we validated these assays and studied the role of O-linked N-acetylglucosamine (O-GlcNAc) modification of NPCs in modulating the kinetics of nuclear import and export. We further expanded the use of these assays for quantitative assessment of pathological proteins that compromise the nucleocytoplasmic transport by combining the assays with a novel technique for measuring intracellular level of exogenously expressed protein. Using this combined assay, we simultaneous measured the intracellular level of SARS-CoV-2 ORF6 protein in transiently transfected cells and the resulting reduction in the nuclear transport kinetics. This enabled a dose-response characterization of the nuclear transport inhibition by ORF6 protein. Using this approach, we quantitatively compared various ORF6 variants, including those of SARS-CoV and SARS-CoV-2, providing a mechanistic insight into how ORF6 inhibits the nuclear transport.