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The surface charge of carbon dots (CDs) governs cellular uptake; however, studies seldom compare CDs with similar physico-chemical properties thereby introducing confounding variables. Here, we investigate how the surface charge of amine-passivated carbon dots with similar optical and chemical properties influences their uptake in human cells. We synthesized CDs using citric acid diethylenetriamine (DT3) or pentaethylenehexamine (PH6) using microwave-assisted synthesis. Extensive characterization confirmed their physico-chemical and optical properties. Ion exchange column chromatography was used to separate CDs into fractions with surface charges ranging from -35 mV to +7 mV, which were then added to HeLa cells. FT-IR analysis shows a visible increase of the amide stretch at 1646 cm^-1 as charge decreases for the separated fractions revealing changes to surface functionalities. Fluorescence microscopy revealed a correlation between surface charge and cellular uptake. Our study shows a greater uptake of DT3-CDs by 1.17-fold with a surface charge of -14 mV, which were also enriched in the cytosol by 4.12-fold, compared to those with a charge of -35 mV, which localized at the lysosomes which is in accordance with our previous study. In contrast, PH6-CDs remained consistent regardless of their charge (+7 mV vs. -6 mV), with a preference for lysosomes. This study reveals how surface charge and chemical composition of CDs impacts cellular uptake and localization. These findings show how CDs could be tailored for specific applications in bioimaging and nanomedicine.