Understanding the cellular uptake mechanism of materials is of fundamental importance that would be beneficial for materials design with enhanced biological functions. Herein, we report the interplay of pharmacological and genetic approaches to minimize the possible misinterpretation on cellular uptake mechanism. A library of amphiphilic polymers was used as a model system to evaluate the reliability of such methodological interplay. To probe the cellular uptake of amphiphilic polymers, we utilized an orthogonal end-group labeling strategy to conjugate one fluorescent molecule on each polymer chain. The results from the methodological interplay with these labeled polymers revealed the off-target effects of dynasore, a well-known dynamin inhibitor. Instead of dynamin, actin was found to be an essential cellular component during the cellular uptake of these amphiphilic polymers. Our study demonstrates the importance of interplaying pharmacological and genetic approaches when evaluating the endocytic mechanism of functional materials, providing insights on understanding the cellular uptake of future therapeutic materials.