Aims: Triple-negative breast cancer patients are commonly treated with combination chemotherapy. Nonetheless, outcomes remain substandard with relapses being of a frequent occurrence. Among the several mechanisms that result in treatment failure, multidrug resistance, which is mediated by ATP-binding cassette proteins, is the most common. Regardless of the substantial studies conducted on the heterogeneity of cancer types, only a few assays can distinguish the variability in multidrug resistance activity between individual cells. We aim to develop a single-cell assay to study this. Methods: This experiment utilized a microfluidic chip to measure the drug accumulation in single breast cancer cells in order to understand the inhibition of drug efflux properties. Results: Selection of single cells, loading of drugs, and fluorescence measurement for intracellular drug accumulation were all conducted on a microfluidic chip. As a result, measurements of the accumulation of chemotherapeutic drugs (e.g., daunorubicin and paclitaxel) in single cells in the presence and absence of cyclosporine A were conducted. Parameters such as initial drug accumulation, signal saturation time, and fold-increase of drug with and without the presence cyclosporine A were also tested. Conclusion: The results display that drug accumulation in a single-cell greatly enhanced over its same-cell control because of inhibition by cyclosporine A. Furthermore, this experiment may provide a platform for future liquid biopsy studies to characterize the multidrug resistance activity at a single-cell level.