Onion (Allium cepa L.) and its newly derived product "black onion" are characterised by the presence of compounds with potential bioactivity, particularly organosulfur compounds (OSCs). However, little is known about the metabolism, distribution, and excretion of these compounds as they pass through the gastrointestinal tract. This study monitored healthy subjects after an acute intake of black onion and analysed the excretion of OSCs using UHPLC-HRMS. A total of 31 OSCs were detected in urine after the acute ingestion of black onion, the main components being S-methyl-L-cysteine sulfoxide (methiin) (13.6 ± 3.9 μmol), isoalliin (12.4 ± 4.7 μmol) and S-propyl-L-cysteine (deoxypropiin) (3.1 ± 0.7 μmol). Moreover, N-acetylated metabolites of the major OSCs detected in black onion, namely, N-acetyl-S-(1-propenyl)-L-cysteine sulfoxide (NAS1PCS) and N-acetyl-S-(1-propenyl)-L-cysteine (NAS1PC), were found in urine after black onion consumption. The N-acetylation reaction takes place in the kidneys and liver, and metabolism pathways are proposed to explain the excretion of OSCs in urine. The basis of the identification of OSCs as urinary metabolites after black onion consumption is described for the first time and provides the basis for further research.