<p>Genetic manipulation of tolerance to herbicides holds a key in timely management of weeds using less labour and cost. Herbicide tolerance in germplasm/segregating population is generally evaluated through destructive method(s) by spraying the herbicide(s) which has the disadvantage of losing precious heterozygous plants. Developing linked molecular markers or laboratory assays would be very much helpful in avoiding those disadvantages. Imazethapyr is a broad-spectrum herbicide inhibits the enzyme acetolactate synthase (ALS) involved in the biosynthesis of amino acids viz., leucine, isoleucine and valine. In this study, we optimized a laboratory protocol to measure the accumulation of acetolactate, a key intermediate metabolite formed by acetolactate synthase (ALS). Functional allele of ALS, insensitive to the herbicide Imazethapyr formed acetolactate which was allowed to react with creatine and naphthol to form the red coloured acetoin. Whereas susceptible allele harbouring plants did not form acetolactate due to the inhibition of ALS by the herbicide Imazethapyr and thus formed pale or yellow colour. This assay effectively distinguished the susceptible genotype ASD 16 from the tolerant mutant Robin-HTM. Laboratory results were validated through greenhouse experiments confirming the susceptibility of ASD 16 and the tolerance of Robin-HTM. These Results pave the way for the rapid, non-destructive evaluation of the introgression or segregation of susceptible/tolerant AHAS/ALS alleles from the herbicide-tolerant rice mutant Robin-HTM, particularly in situations where gene-based markers are unavailable for tracking herbicide tolerance.</p>