In this work, a new method employing capillary electrophoresis (CE) for the determination of several species in biodiesel is introduced. The concentrations of inorganic species (Na+, K+, Ca2+, Mg2+, SO42−, and PO43−) and glycerol are of interest to the regulatory authorities due to their ability to form undesirable compounds in engines. Additionally, other species of low molecular weight (e.g., acetate, formate, and propionate) are of interest because they contribute towards increasing the acidity. These species are formed by the degradation of biodiesel and cause damage to engines and the environment. The cation separation was performed in background electrolyte (BGE) composed of 30mmolL−1 of 2-(n-morpholino)ethanesulfonic acid (MES)/L-histidine (His), pH 6. The separation of anionic species was carried out in similar BGE with 0.2mmolL−1 cetyltrimethylammonium bromide (CTAB) added. For glycerol, a neutral species, its oxidation with periodate was employed. This well-known reaction is specific to polyols and generates iodate. The amount of iodate produced by the reaction was determined by CE. The separation was carried out in approximately 1min using BGE composed of 30mmolL−1 acetic acid, pH 3. The analytical parameters evaluated were: linearity (r>0.99), the RSD values for area and migration time were