Cellular uptake pathway of nanoparticle (NP) is different from that of free drugs. Therefore, NP-mediated nanotherapeutics can be designed to overcome the adverse effects of free drugs. However, synthetic NPs are typically trapped in the endosome and have difficulty to reach the cytosol because of the characteristic endocytosis, where the endosomal membranes wrap-up the introduced NPs. In this study, the Spacer molecules linking the apoptotic anticancer drug and the gold NP (AuNP) are designed and cellular uptake procedure and drug deployment in the cancer cells are controlled. X-ray nanoscopy and two-photon microscopy are employed to observe the AuNPs in a cell in-situ without additional dye molecule or imaging agent introduction on an AuNP. We confirm that the effective design of the Spacer molecules importantly control the cellular interaction of the AuNPs. This technology can be generalized to broad biomedical applications utilizing nanotherapeutics-mediated diagnosis and new-concepted disease treatment technologies.