© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Biodiesel production from the transesterification of triglycerides produces crude glycerol as a by-product with a percentage of glycerol typically 20–80% (w/w) depending on the specific conditions of the transesterification process. This crude glycerol requires further purification in order to achieve commercial value and to increase the profitability of biodiesel production. For this reason, the main objective of this work was to obtain glycerol with a purity greater than 90% (w/w) starting from water-free crude glycerine as obtained from the IPN-GBD-1000® transesterification process and treating it via single-step neutralization according to green chemistry principles. For this purpose, sulphuric (H2SO4) and citric (C6H8O7) acids were evaluated as neutralizers by adding dilute acid solutions to crude glycerine under mild conditions. The physicochemical characterization of both crude and purified glycerol was carried out by means of infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (NMR) and thermogravimetric analysis (TGA). The results indicated that the neutralization method herein developed allowed the obtaining of glycerol with purities of 98.5% and 84.37% (w/w) and treatment efficiencies of 98.5% and 46.7% for sulphuric and citric acid treatments, respectively. In addition, the environmental viability of the sulphuric acid process was evaluated through the calculation of green metrics such as environmental factor, water factor and mass intensity, through which significant environmental advantages were confirmed. The one-step neutralization process reported herein generates zero waste when sulphuric acid is used; it also decreases the water consumption 17-fold and reduces 3-fold the use of raw materials per mass unit of purified glycerol compared to the conventional acidification-neutralization process.