Abstract: Over recent decades, sulfur fumigation has been becoming abused in processing some fresh Chinese herbal medicines due to its benefits containing simple operation, declined cost, and bleaching color characteristics. However, it has recently emerged as a controversial topic with respect to the potential detrimental effects, such as chemical transformation of inherent constituents and consequently the generation of potential toxicities. Radix Ophiopogon, the root of Liliaceae Ophiopogon japonicus (Thunb.) Ker Gawl, is a kind of traditional Chinese medicine that always suffers sulfur fumigated (SF). The modern phytochemistry researches have demonstrated that homoisoflavones, steroidal saponins and polysaccharides are the dominant components of this Chinese medicine. Although several researches have focused on the sulfur fumigated Ophiopogon japonicus till now, there was no study to reveal the phenomenon of chemical transformation of inherent constituents. Herein, in this study, the new generated homoisoflavones sulfur derivatives were researched to preliminarily clarify its transformation pathways during sulfur fumigation process based on UHPLC-LTQ-Orbitrap mass spectrometry. The separation was conducted on a Waters Acuqity UPLC HSS T3 C18 column with the gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile/methanol (3∶1, V/V) (B). To reduce interference of other substances and avoid the missing of the trace sulfur derivatives, the parent ion list-dynamic exclusion in negative mode based data-acquisition method was established. As a result, 41 homoisoflavones sulfated derivatives are detected. Among them, 31 are identified based on the high-resolution MS/MS datasets. This study tentatively clarified the transformation pathways of homoisoflavones during sulfur fumigation, and provided some useful information for the following relevant studies.