The next generation of therapeutic nanoparticles in the treatment of cancer incorporate specific targeting. There is implicit importance in understanding penetration of targeted nanomedicines within tumour tissues via accurate and quantitative temporospatial measurements. In this study we demonstrate the potential of state-of-the-art synchrotron X-ray fluorescence microscopy (XFM) to provide such insights. To this end, quantitative mapping of the distribution of transferrin-conjugated gold nanoparticles inside multicellular tumour spheroids was achieved using XFM and compared with qualitative data obtained using reflectance confocal microscopy. Gold nanoparticles conjugated with human transferrin with a narrow size-distribution and high binding affinity to tumour cells were prepared as confirmed by cellular uptake studies performed on 2D monolayers. Although the prepared 100 nm transferrin-conjugated gold nanoparticles had high targeting capability to cancer cells, penetration inside multicellular spheroids was limited even after 48 hours as shown by the quantitative XFM measurements. The rapid, quantitative and label-free nature of state-of-the-art synchrotron XFM make it an ideal technology to provide the structure-activity relationship understanding urgently required for developing the next generation of immuno-targeted nanomedicines.