An optimal design of nanoparticles suitable for biomedical applications requires proper functionalization, a key step in the synthesis of such nanoparticles, not only for subsequent crosslinking to biological targets and to avoid cytotoxicity, but also to endow these materials with colloidal stability. In this sense, a reliable characterization of the effectiveness of the functionalization process would, therefore, be crucial for subsequent bioconjugations. In this work, we have analyzed glutathione as a means to functionalize four of the most widely used nanoparticles in biomedicine, one of which is a hybrid gold-magnetic-iron-oxide nanoparticle synthetized by a simple and novel method that we propose in this article. We have analyzed the colloidal characteristics that the glutathione capping provides to the different nanoparticles and, using information on the Z-potential, we have deduced the chemical group used by glutathione to link to the nanoparticle core. We have used electron microscopy for further structural and chemical characterization of the nanoparticles. Finally, we have evaluated nanoparticle cytotoxicity, studying cell viability after incubation with different concentrations of nanoparticles, showing their suitability for biomedical applications.