A sensitive and selective spectrophotometric method for the estimation of the toxic factors mimosine and 3-hydroxy-4-(1H)-pyridone (DHP) has been developed based on the intense yellow-colored azodye formed with p-nitroaniline which showed a sharp absorption maximum at 400 nm. The method was optimized based on relative sensitivity of the reaction with various aromatic primary amino compounds and under different conditions of pH. Interference from a variety of structurally related compounds and phenols was tested and found insensitive to this method. The molar extinction coefficient at 400 nm for the azodye formed with mimosine was 5.31 × 104 M-1 cm-1 and that for DHP was 1.699 × 104 M-1 cm-1. The applicability of the method was tested using different plant extracts and recovery was found to be at 100 ± 0.3%. The method is suitable for accurate estimation of both mimosine and DHP after paper chromatographic separation of extracts of different biological samples. © 1993 Academic Press. All rights reserved.