The Pig-a gene is well-known to encode a key enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI), which attaches CD molecules (Cluster differentiation), such as CD55 and CD59, to red blood cells (RBCs) membranes. In this study, the blood was marked with the special antigen CD45-PE (Phycoerythrin) to separate the erythrocytes from the leukocytes, then the Pig-a mutant frequency (MF) of RBCs could be investigated without pivotal antigen CD59-FITC (Fluorescein isothiocyanate), and the optimal ENU concentration was determined to be 100 mg/kg/day. The optimal cell number to count and the stability of specimens were determined. At last, the in vivo Pig-a gene mutation assay was utilized to detect the potential genotoxicity of Cis-Dichlorodiammineplatinum (DP), Procarbazine Hydrochloride (PH), and Triptolide (TP). The results indicated that the Pig-a gene mutation obviously occurred in the PH treatment group. In the DP treatment group, an irregular shape with a slightly serrated border in erythrocytes was observed in the blood smear. However, no obvious Pig-a gene mutations were detected in the DP treatment and TP treatment groups under the experimental conditions. In conclusion, this primary in vivo Pig-a gene mutation assay will provide an easy protocol to use in screening potential genotoxic compounds.