Penetration of nanoparticles into viable tumor regions is essential for an effective response. Mass spectrometry imaging (MSI) is a novel method for evaluating the intratumoral pharmacokinetics (PK) of a drug in terms of spatial distribution. The application of MSI for analysis of nanomedicine PK remains in its infancy. In this study, we evaluated the applicability of MALDI-MSI for nanoparticle-formulated drug visualization in tumors and biopsies, with an aim toward future application in clinical nanomedicine research. We established an analytic method for the free drug (AZD2811) and then applied it to visualize nanoparticle-formulated AZD2811. MSI analysis demonstrated heterogeneous intratumoral drug distribution in three xenograft tumors. The intensity of MSI signals correlated well with total drug concentration in tumors, indicating that drug distribution can be monitored quantitatively. Analysis of tumor biopsies indicated that MSI is applicable for analyzing the distribution of nanoparticle-formulated drugs in tumor biopsies, suggesting clinical applicability.