Heat shock factors (HSFs) are vital for modulating stress and heat shock-related gene expression in cells. The activity of HSFs is controlled largely by post-translational modifications (PTMs). For example, basal phosphorylation of HSF1 on three serine sites suppresses the heat shock response, and hyperphosphorylation of HSF1 on several other serine and threonine sites by stress-activated kinases results in its activation, while acetylation on K80 inhibits its DNA-binding ability. Sumoylation of HSF2 on K82 regulates its DNA-binding ability, whereas sumoylation of HSF4B on K293 represses its transcriptional activity. With the advancement of proteomic technology, novel PTM sites on various HSFs have been identified with the use of tandem mass spectrometry (MS/MS), but the functions of many of these PTMs are still unclear. Yet, it should be noted that the discovery of these novel PTM sites provided the necessary evidence for the existence of these PTM marks in vivo. Followed by subsequent functional analysis, this would ultimately lead to a better understanding of these PTM marks. MS/MS-based proteomic approach is becoming a gold standard in PTM validation in the field of life science. Here, the recent literature of all known PTMs reported on human HSFs and the resulting functions will be discussed.