A posttranslational modification that is unique for the protein tubulin is tyrosination/detyrosination of the C-terminus of α-tubulin. Detyrosination of the genetically encoded C-terminal tyrosine by an unidentified carboxypeptidase is followed by retyrosination catalyzed by the enzyme tubulin tyrosine ligase (TTL). Proper functioning of tyrosination/detyrosination cycle is required for cell vitality. In particular, TTL downregulation and increase of detyrosinated tubulin correlate with increased tumorigenesis and tumor invasiveness. Exogenous modulators of TTL may restore tyrosinated tubulin and impair tumor progression. The only method currently available for studying such modulators requires use of radioactive ligands. Here, we present an assay suitable for high- throughput screening of TTL effectors, in which the readout is fluorescence- or immunochemistry- based. In this assay, detyrosinated tubulin and TTL are allowed to react with a potential modulator in the presence of 3-formyltyrosine (3fY), a known substrate for TTL. The ligand's ability to compete with 3fY is quantified by the fluorescence signal generated when the protein bound aldehyde group covalently reacts with a suitably derivatized fluorophore. In addition, we have established an immunochemical detection method in which biotin hydrazide is used instead of a fluorophore. The extent of biotinylation is then detected using Streptavidin-HRP conjugate. Both these detection methods generate comparable results. Thus, we have developed a versatile, sensitive, non-radioactive high- throughput screening assay that can be employed to examine TTL modulators.