The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., 68ZnSO4), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.