Degumming is an oil refinement process in which the naturally occurring phospholipids in crude vegetable oils are removed. Enzymatic degumming results in higher oil yield and more cost-efficient processing compared to traditional degumming processes using only water or acid. Phospholipase C hydrolyses phospholipids into diglycerides and phosphate groups during degumming. The diglyceride content can therefore be considered a good indicator of the state of the enzymatic reaction. This study investigates the use of near-infrared (NIR) spectroscopy and chemometrics to monitor the degumming process by quantifying diglycerides in soybean oil in both off-line and on-line settings. Fifteen enzymatic degumming lab scale batches originating from a definitive screening design (with varying water, acid, and enzyme dosages) were investigated with the aim to develop a NIR spectroscopy prediction method. By applying tailored preprocessing and variable selection methods, the diglyceride content can be predicted with a root mean square error of prediction of 0.06% (w/w) for the off-line set-up and 0.07% (w/w) for the on-line set-up. The results show that the diglyceride content is a good indicator of the enzyme performance and that NIR spectroscopy is a suitable analytical technique for robust real-time diglyceride quantification.