AbstractDealing with nude mice, which lack thymus and therefore are sensitive to unsterile conditions, needs special care and laboratory conditions. For preclinical studies, especially tumour imaging purposes, in which therapeutic properties of drugs or therapeutic compounds are not studied, mice with normal immune system can be a favourable alternative if they carry tumours of interest. In the current study, we introduce an optimized protocol for induction of human tumours in BALB/c mice for preclinical studies. Immune system of BALB/c mice was suppressed by administration of cyclosporine A (CsA), ketoconazole and cyclophosphamide. The tumours of MDA‐MB‐231, A‐431 and U‐87‐MG human cancer cells were induced by subcutaneous injection of the cells to the immunosuppressed mice. Tumour size was calculated weekly. Histopathological and metastatic analyses were performed using haematoxylin and eosin staining. The combination of the three drugs was found to suppress immune system and decrease the numbers of white blood cells, including lymphocytes. At the eighth week, tumours with a dimension of approximately 1400 mm3developed. Large atypical nuclei with scant cytoplasm were found to exist using histopathological analysis. No metastasis was observed in the tumour‐bearing mice. A combination of CsA, ketoconazole and cyclophosphamide can be used to suppress the immune system in BALB/c mice and induce tumours with significant size.