HS donors are substitutes of HS with various biological activities like inhibiting the inflammatory response and protecting myocardial cells from injury. In order to confirm whether the HS donors have drug-like properties, two series thiophosphamide HS donors were evaluated including toxicity, bioactivity and pharmacokinetic properties in vivo and in vitro. The following results were obtained. Firstly, all the compounds released HS under measuring condition; with the increase of pH value, the HS release rate of all the compounds decreased and the amount reduced, but pH value had little effect on the maximum release of HS. Secondly, in the organs and tissues of rats, the compounds released HS in the same way as in PBS. In plasma, compound 1 reached the C after administration 55 min, and no compound 1 was detected after 12 h; for compound 18, the C reached only after administration 100 min, and after 6 h, compound 18 was not detected; in organs and tissues, the HS-release rates were different from those in PBS, but the mechanism of HS release was the same. Thirdly, in the test of toxicity, all the compounds displayed low toxicities to 5 cancer cells and W138 cell lines; compounds 1 and 18 had slight effect on the physiological tissue and function of rat liver at low concentration; the compounds had almost no effect on the hatching rate, survival rate of zebrafish embryos, and the spontaneous movement of zebrafish embryos at below 0.5 μM, but when they were over 1 μM, the compounds displayed inhibitory effect in the manner of concentration dependence. Fourthly, in the course of anti-inflammatory test, all the tested compounds significantly reduced the level of TNF-α and increased the level of IL-10; when they were 100 μM, the levels of IL-10 were three times as high as those in the control group. Among them, compounds 10 and 18 displayed stronger activities than the others. In addition, the compounds protected H9c2 cells from injure and improved myocardial injury through anti-oxidation pathway. In summary, the compounds have druglike properties due to low toxicity, better activity and good pharmacokinetic property. Therefore, they have potential to be as candidates to investigate further.