Methanol quenching and fast filtration, the two most common sampling protocols in microbial metabolome analysis, were validated for intracellular amino acid analysis in phylogenetically different yeast strains comprising Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia pastoris, Schizosaccharomyces pombe and Zygosaccharomyces bailii. With only few exceptions for selected amino acids, all yeasts exhibited negligible metabolite leakage during quenching with 60% cold buffered methanol. Slightly higher leakage was observed with increasing methanol content in the quenching solution. Fast filtration resulted in identical levels for intracellular amino acids in all strains tested. The results clearly demonstrate the validity of both approaches for leakage-free sampling of amino acids in yeast.