Efficient cell internalization of framework nucleic acid nanostructures free of transfection agents provides new opportunities for developing biocompatible and intelligent nanoprobes and drug delivery carriers. Here, a proteomic identification method to screen target proteins that interact with tetrahedral DNA nanostructures (TDNs) during the process of endocytosis by combining drug affinity responsive target stability (DARTS) with liquid chromatography/tandem mass spectrometry (LC-MS/MS) techniques, is reported. It is found that that caveolin-1 (CAV1) and macropinocytosis-related protein sorting nexin5 (SNX5) are associated with the endocytosis of TNDs, which is further validated by microscale thermophoresis (MST) analysis. CAV1- and SNX5- knockout experiments reveal that both caveolae-mediated endocytosis and macropinocytosis mediate the cellular uptake of TDNs, which complement previous findings with fluorescence tracing methods. This method provides a generic strategy to analyze cellular internalization process of DNA nanostructures for biomedical applications.