For the screening of yeast transketolase (TK) variants with improved or new properties acquired by random mutagenesis, we report on the stereoselective synthesis of fluorogenic substrates as probes for measuring TK activity. Compound 1 (7-(2′,3′,5′-trihydroxy-4′-oxo-pentyl)oxycoumarine), prepared as previously described, [Tetrahedron Lett. 2003, 44, 827–830] enabled us to evaluate wild type TK velocity in a simple, specific and reproducible way. To select TK mutants able to produce d-threo aldoses, we prepared compound 2 (dihydroxy-4-O-(2′-oxo-benzopyran-7′-yl-D-threose) from dimethyl tartrate. Starting from d-ribose, we successfully obtained compound 3 (7′-(2,3,5-trihydroxy-4-oxo-pentyl)oxycoumarine) as a probe for TK mutants able to produce l-erythro ketoses.