This study presents comparison of the immunosuppressive effects of Azathioprine (AZA) and Cyclophosphamide (CPM) in CD-1® mice and to generate positive control data. The mice were treated with the AZAat the dose level of 20 and 50 mg/kg b. wt. and CPM at the dose level of 20mg/kg b. wt.for 4 consecutive days. Control group animals were treated with RO water. All the animals showed no clinical signs throughout observation period of experiment. Blood samples were collected at termination and analysed for Lymphocytes’ subpopulations and NK cells by FACSVerseTM. Iimmunosuppressive effects on T cells (CD3+, CD4 and CD8), B cells (CD19, CD45) and natural killer cells (CD3-, CD16) were significant in male and female mice treated with CPM at the dose level of 20 mg/kg b. wt and AZA at 50 mg/kg b. wt. The effects in both the groups were identical. Based on these results, it could be concluded that CPM at 20 and AZA at 50 mg/kg b. wt. found to have similar immunosuppressive effects in CD-1®mice. Female mice could be considered more sensitive than male as both helper and cytotoxic T cells (CD4- and CD8) suppressed by CPM at 20 and AZA at 50 mg/kg b. wt. However, further experimental evaluations should confirm the findings in respect of sex sensitivity. Results of this study will be helpful for comparative assessment of lymphocytic enumeration in the future studies.Keywords: FACSVerseTM (Fluorescence Activated Cell Sorting), Cluster of Differentiation (CD) cells