XCT, a well-known cystine transporter, is reported to be involved in the proliferation of various cells, such as cancer cells, immune cells, and fibroblasts. xCT inhibitor is expected to be a promising drug for cancer or immune diseases. However, there are little studies reporting that xCT inhibitors improve disease progression in vivo. To invent potent xCT inhibitors in vivo, we established a new in vivo model for assessing efficacy of xCT inhibition.DL-propargylglycine (PPG) was administered i.p. to wild-type C57BL/6J mice. Concentration of cystathionine, another substrate of xCT, in the thymus and spleen was measured by LC-MS/MS.PPG increased cystathionin amounts in the thymus and spleen in a dose- and time-dependent manner. At 7 h after PPG administration, the efficacy of erastin, a representative xCT inhibitor, was clearly shown. This paper synthesized a new compound, compound A, which had much higher inhibitory effect on xCT than erastin both in vitro and in vivo.This paper established a mouse model of PPG-induced cystathionine accumulation for assessing xCT inhibition in vivo. By using this model, we discovered that compound A was approx. 15 times more effective in vivo than erastin.