Glycerol, a waste product of biodiesel production, currently has a cost associated with its disposal. Complete glycerol oxidation to CO₂ in acid medium is difficult to achieve, but an alkaline medium facilitates this process. It is known, that non-noble metals such as Cu, Ni, Fe and others, decrease the CO-poisoning effect. Pt is a promising electrocatalyst for alkaline medium, however, it requires co-catalysts to enhance the oxidation process. In this work, we evaluate the effect of Ni in PtNi catalyst toward glycerol electro-oxidation by two methods: cyclic voltammetry and in-situ micro Raman Spectroscopy. For this reason, metal nanoparticles of Pt and PtNi were synthesized using Vulcan Carbon as support. According with XRD, crystallite sizes of 4.48 and 4.20 nm were obtained for Pt/C and PtNi/C, respectively. TEM micrographs showed semispherical-shaped nanoparticles with sizes closed to their crystallite sizes. For metallic mixture, X-ray fluorescence revealed metallic composition of Ni₆₅Pt₃₅. Moreover, thermogravimetric analysis showed a metal mass content of 3 wt. % for Pt/C and 18 wt.% for PtNi/C. The electrocatalytic activity toward glycerol electro-oxidation was tested as function of its concentration, variation of nature of the supporting electrolyte and the electrolyte concentration. Pt and PtNi/C showed oxidation potentials of 0.174 and 0.10 V vs. NHE at 0.5M glycerol, respectively. Also, the i_f/i_b rate as parameter for CO-tolerance, indicated that PtNi/C exhibited a higher tolerance with a 3.5-fold higher rate; meanwhile, Pt/C showed a 2.1 rate.