The aim of the present work was to develop and evaluate near-infrared (NIR) and fluorescence spectroscopies as rapid and potential online methods for determination of the amount of aleurone in pearling dust fractions of barley. Phytic acid was used as a marker for the aleurone layer. Different varieties of barley were pearled, and dust fractions were progressively taken out. Sample concentration of phytic acid varied in the range of 0.5-4.1 g/100 g, and highest concentrations were obtained in dust fractions for pearling degrees in the range of 15-25%. Regression models for phytic acid were developed with the same high correlations for NIR and fluorescence spectroscopies (R2 = 0.96) and prediction errors of ±0.16-0.18 g/100 g. The models performed well on a test set of pearling fractions from two other barley varieties. The techniques are rapid and nondestructive, which means that they can be used online in connection with industrial pearling systems.