Glycerol (GlOH) accumulation and its very low price constitute a real problem for the biodiesel industry. To overcome these problems, it is imperative to find new GlOH applications. In this context, electrochemistry arises as an important alternative to the production of energy or fine chemicals using GlOH as a reactant. To make these opportunities a reality, it is fundamentally necessary to understand how the glycerol electro-oxidation reaction (GEOR) occurs on catalysts used in real systems. Thus, research using model surfaces has generated the first insight into the electrochemistry of extremely complex real catalysts. Accordingly, in this work, we generate Pt(100) disturbed surfaces in a reproducible manner, carefully controlling the surface defect density. Then, GEOR is studied on well-ordered Pt(100) and on the disturbed Pt(100) surfaces in 0.5 M H₂SO₄ using cyclic voltammetry (CV) and in situ Fourier transform infrared spectroscopy (FTIR). The CV profile of GEOR consists of a single peak in the positive scan. The onset reaction displays the influence of defects present on the surface. On a surface with a high degree of disorder, the main GlOH oxidation process begins at 0.8 V vs RHE, whereas for well-ordered Pt(100), it starts 0.1 V earlier. FTIR experiments show the presence of carbon monoxide and carbonyl absorption bands. The electrochemical and spectroelectrochemical results are supported by density functional theory calculations showing that both CO and GlOH bind more strongly on disturbed than on well-ordered surfaces. Thus, our experiments show that Pt–CO (or other GlOH residue) bond breaking may be the GEOR rate-determining step.