Prostate cancer (PCA) is the most common cancer and the third most common cause of cancer death in men. Targeted nanoparticles (NPs) that deliver effective doses of chemotherapeutic drugs specifically to PCA could improve chemotherapy efficacy without the toxicities. In the relevant mouse models, the direct visualization of such drug nanoparticles along with the vasculature and fibrillar collagen matrix at submicron resolution are critically important for the accurate measurements of the drug distribution in the tissue matrix. Multiphoton microscopy, which uses ultra-short IR laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from exogenous or endogenous fluorescent proteins and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens. The objective here is to visualize and quantify the 3D distribution of an aptamer conjugated calcium phosphosilicate based drug nanoparticle carriers along with vasculature and tissue matrix in ex vivo thick mouse prostate tumor tissue with submicron resolution. Human prostate tumor xenografts were established in athymic mice by injecting prostate cell line derived from human (PC-3 cells) and were grown for 4 weeks. Near-infrared imaging agent indocyanine green (ICG) loaded calcium phosphosilicate nanoparticles (CPSNPs) including targeted CPSNPs bioconjugated with DNA Aptamer, empty non-ICG containing CPSNPs (Ghost) and Dil (for blood vessel painting) were injected into the tail vein. The spectral unmixing was performed to extract Dil signal from ICG signal using measured emission spectra. The 3D reconstructions and subsequent quantitation showed accumulation of ICG in blood capillaries versus tissue matrix. We here conclude that this multiphoton based multimodal imaging approach can provide spatially resolved 3D images with spectral specificities that are sensitive enough to identity and quantify the distributions of drug nanoparticle carriers in conjunction with vasculature and tissue matrix in prostate tumor with structural precision.