The aim of the present work is to investigate, under lab-scale conditions, the removal and transformation of the antibiotic sulfapyridine (SPY) upon advanced oxidation with UV/H₂O₂. High resolution mass spectrometry (HRMS) analyses by means of an ultra-high pressure liquid chromatography (UHPLC)-linear ion trap high resolution Orbitrap instrument (LTQ-Orbitrap-MS) were carried out in order to elucidate the different transformation products (TPs) generated. The abatement (>99%) of the antibiotic was only achieved after 180 min, highlighting its resilience to elimination and its potential persistence in the environment A total of 10 TPs for SPY were detected and their molecular structures elucidated by means of MS² and MS³ scans. Finally, the combined ecotoxicity at different treatment times was evaluated by means of bioluminescence inhibition assays with the marine bacteria Vibrio fischeri.