AbstractHydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method for the determination of hydrogen sulfide in various biological matrices by determination of a derivative of hydrogen sulfide and monobromobimane named sulfide dibimane (SDB). ³⁶S-labeled SDB was synthesized and validated for using as an internal standard. This method has been successfully used to measure hydrogen sulfide levels in a broad range of biological matrices, such as blood, plasma, tissues, cells, and enzymes, across different species. Moreover, a novel mode that hydrogen sulfide could loosely and non-covalently bind to human serum protein (HSA) and hemoglobin (HB) was revealed by using the developed method.