Near-infrared (NIR) spectroscopy was evaluated as a rapid method of predicting arachidonic acid content in powdered oil without the need for oil extraction. NIR spectra of powdered oil samples were obtained with an NIR spectrometer and correlated with arachidonic acid content determined by a modification of the AOCS Method. Partial Least-Squares regression was applied to calculate models for the prediction of arachidonic acid. The model developed with the raw spectra had the best performance in cross-validation (n = 72) and validation (n = 21) with a correlation coefficient of 0.965, and the root mean square error of cross-validation and prediction were both 0.50. The results show that NIR, a well-established and widely applied technique, can be applied to determine the arachidonic acid content in powdered oil.