No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4′-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4′-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4′-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solutionphase transfer of either biotin- or fluorescein-labeled 4′-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4′-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, highthroughput method to determine PPTase activity.