Donor haematopoietic stem cell transplantation (HSCT) is a curative therapy for leukaemia and seeks to induce the graft-versus-leukaemia (GVL) effect, where the donor T cells destroy any remaining malignant cells. This study aimed to determine whether post-transplant cyclophosphamide (PTCy), a treatment for graft-versus-host disease, impacts the GVL effect in a humanised mouse model. To establish the GVL model, 10 million human peripheral blood mononuclear cells (hPBMCs) from three donors or saline were injected i.p. into NSG mice on Day 0, followed by intravenous injection of 1 million human THP-1 leukaemia cells on Day 14. In a subsequent experiment, PTCy was injected at Days 3 and 4 post-injection of 20 million hPBMC. Mice were monitored for clinical signs of disease over 42 days. At endpoint, tissues were collected and human cell engraftment in the liver and spleen was analysed via flow cytometry. Flow cytometry revealed that 1 million THP-1 cells, injected in the absence of hPBMCs, caused leukaemia (hCD45+ cells) in the livers of NSG mice. Mice injected with hPBMCs from different donors and THP-1 cells showed similar survival, clinical score and weight loss. Similar proportions of hCD33+ leukaemia cells were observed in mice injected with THP-1 cells alone and hPBMCs from one donor with THP-1 cells. In contrast, hCD33+ cells were reduced in mice that received hPBMCs from the other two donors, suggesting a donor-specific GVL effect. In subsequent experiments, PTCy-treated humanised mice demonstrated prolonged survival (MST, 40 days) compared to control (MST, 34 days). Notably, both groups showed no hCD33+ cells in the liver, indicating PTCy does not impact GVL responses. This study outlines the establishment of a GVL model in humanised mice, which can be used to test therapies that prevent GVHD but retain GVL responses.