Biodiesel has recently gained importance as an alternative to fossil diesel. However, the development of more efficient biodiesel formation processes still depends on the use of fast and accurate analytical techniques to evaluate the conversion degree of the transesterification reaction. Nuclear magnetic resonance (NMR) spectroscopy has been used for this purpose, but some experimental details still need to be addressed. Therefore, in this communication, the experimental conditions for a truly quantitative NMR analysis of biodiesel formation are presented. The longitudinal relaxation time (T1), which is the determining factor for quantitative analysis, was measured using an inversion-recovery method, and a maximum value of 2.35 s was obtained for a biodiesel sample. A linear determination coefficient of r2 = 0.99 was obtained when a time delay between pulses longer than 5T1 =15 s was used, whereas strong deviations were observed when using shorter delays. Copyright © 2012 John Wiley & Sons, Ltd.