A team of experts from twelve countries set up by the EU Commission`s DG XII Measurements & Testing Programme has devised an optimal analytical method for the determination of tryptophan in feedstuffs by conducting three comparative inter-laboratory tests. The influence of the hydrolysis reagent (barium hydroxide, lithium hydroxide or sodium hydroxide) and the apparatus used, of various ways of incorporating an internal standard and of the method of detection on tryptophan recovery and reproducibility of the results was investigated and extraction conditions for supplemented tryptophan were elaborated. In a unique collaborative approach all participants tested and compared different measurement variants. The results were significant and allowed firm conclusions to be drawn about the optimal analytical conditions. The best results were achieved with protein hydrolysis using barium hydroxide in an autoclave, followed by the addition of the internal standard alpha-methyl-tryptophan and HPLC with fluorescence detection. Extraction with diluted hydrochloric acid with the addition of alpha-MT as internal standard provides reliable results for free tryptophan, although allowance must be made for native blank values. The procedure has been submitted for adoption as the candidate official EU method and has been used for characterising four candidate standard reference materials