The metabolome, the entirety of the small molecules produced by an organism, represents a vast and still incompletely characterized world of chemical structures that serve a myriad of different biological functions. To understand mechanisms by which the abundance of metabolites, and thus their associated functions, are regulated, the elucidation of their biosynthetic pathways is of great importance. However, structural characterization of biosynthetic intermediates is challenging, because they often represent reactive electrophilic species that decompose during conventional extraction and sample preparation procedures. Due to the lack of viable methods for the characterization of such biosynthetic intermediates, the biosynthetic steps involved in the production of many important metabolites have remained unclear. In this study, a newly developed hydroxylamine-derived probe that can trap transient electrophilic biosynthetic intermediates was used to investigate the biosynthesis of gliotoxin, a fungal secondary metabolite derived from a non-ribosomal peptide synthetase (NRPS) pathway in the human pathogen Aspergillus fumigatus. Gliotoxin is a diketopiperazine (DKP) derived from a modified dipeptide; however, the exact sequence by which the precursor dipeptide is converted into gliotoxin remains unclear. To obtain more detailed insight in gliotoxin biosynthesis, we employed TAMOHA (4-aminooxy-N,N,N-trimethylbutan-1-aminium), a hydroxylamine derivative that was recently developed in the Schroeder lab to trap electrophilic intermediates such as transiently stable thioesters as stable hydroxamic acids. In addition to a hydroxylamine moiety, the structure of TAMOHA contains a constitutively positively charged quaternary ammonium terminus, which enhances detection of the resulting hydroxamic acids via mass spectrometry. In this study, we first synthesized a sample of TAMOHA via a three-step sequence using the Gabriel amine synthesis. Next, by using TAMOHA to trap thioester intermediates of the Gliotoxin pathway, we identified the structures of previously uncharacterized biosynthetic intermediates, providing key insights into the sequence of steps converting a simple dipeptide into gliotoxin. The identified structures were elucidated based on the analysis of high-resolution mass spectra and MS/MS fragmentation data. This study demonstrates the potential of TAMOHA to identify electrophilic intermediates in diverse metabolic pathways.