Our observations on the growth stimulatory nature of mimosine, (β-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15-35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing Rf, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 μM or DHP from 0.2 to 1.5 μM and with recoveries of 99.2 to 100.5%. © 2006 Springer-Verlag.