S-Persulfidation generates persulfide adducts (RSSH) on both small molecules and proteins. This process is believed to be critical in the regulation of biological functions of reactive sulfur species such as H2S, as well as in signal transduction. S-Persulfidation also plays regulatory roles in human health and diseases. Recent Advances: Some mechanisms underlying the generation of low molecular weight (LMW) persulfides and protein S-persulfidation in living organisms have been uncovered. Some methods for the specific delivery of persulfides and the detection of persulfides in biological systems have been developed. These advances help to pave the road to better understand the functions of S-persulfidation.Persulfides are highly reactive and unstable. Currently their identification relies on trapping them by S-alkylation but this is not always reliable due to rapid sulfur exchange reactions. Therefore the presence, identity, and fates of persulfides in biological environments are sometimes difficult to track.Further understanding the fundamental chemistry/biochemistry of persulfides and development of more reliable detection methods are needed. S-Persulfidation in specific protein targets is essential in organismal physiologic health and human disease states. Besides cardiovascular and neuronal systems, the roles of persulfidation in other systems need to be further explored. Contradictory results of persulfidation in biology, especially in cancer, need to be clarified.