Due to the cellular heterogeneity in multidrug resistance (MDR) cell populations, positive drug effects on the modulation of MDR can be obscured in conventional methods, especially when only a small number of cells are available. To address cellular variations among different MDR cells, we report a new microfluidic approach to study drug effect on MDR modulation, by investigating drug accumulation of daunorubicin in MDR leukemia cells. We have demonstrated that the new approach of same-single-cell analysis by accumulation (denoted as SASCA-A) is not only superior to different-single-cell analysis, but also has key advantages over our previous approach of same-single-cell analysis. First, SASCA-A is much simpler as it does not require multiple cycles of drug uptake and drug efflux. Second, it is faster, only taking about one fourth of the time used in the previous approach. Third, it provides a more 'identical' and reliable control because it compares the time points just before MDR modulator tests. To help understand the dynamics of drug accumulation in MDR cells, we also developed a mathematical model to describe the kinetics of drug accumulation conducted in individual cells. The SASCA-A method will benefit drug resistance research in minor cell subpopulations (e.g., cancer "stem" cells) because this method requires only a small number of cells in identifying the MDR reversal effect.