Gold nanoparticles are prime candidates for cancer thermotherapy. However, while the ultimate target for nanoparticle-mediated photothermal therapy is the cancer cell, heating performance has not previously been evaluated in the tumoral environment. A systematic investigation of gold nanostar heat-generating efficiency in situ is presented: not only in cancer cells in vitro but also after intratumoral injection in vivo. It is demonstrated that (i) in aqueous dispersion, heat generation is governed by particle size and exciting laser wavelength; (ii) in cancer cells in vitro, heat generation is still very efficient, but irrespective of both particle size and laser wavelength; and (iii) heat generation by nanostars injected into tumors in vivo evolves with time, as the nanostars are trafficked from the extracellular matrix into endosomes. The plasmonic heating response thus serves as a signature of nanoparticle internalization in cells, bringing the ultimate goal of nanoparticle-mediated photothermal therapy a step closer.