INTRODUCTION: Flow cytometry is highly sensitive for detection and quantitative analysis of surface and intracellular antigens in malignant hemopoieticcells and is used for classification and lineage assignment of acute leukemia. AIM– To identify the role of a single 5 colour, CD45, myeloperoxidase (MPO), cCD79a, cCD3, and Tdt, cytoplasmic markers combination as a primary tube and comparison with final diagnosis done on the basis of morphology and primary and secondary panels in immunophenotyping. MATERIALS AND METHODS: We have included 200 new cases of pediatric acute leukemia. We analyzed sensitivity and specificity of different subsets with combination of positive and negative markers. RESULTS: MPO was positive in 76.9% of acute myeloid leukemia (AML) cases. All 39 (100%) cases of the AML were negative for cCD3 andcCD79a co‑expression. cCD79a expression was highly sensitive as 97.7% B‑acute lymphoblastic leukemia (B‑ALL) expressed it. cCD3 expression was detected in 100% cases of T‑ALL, and its co‑expression was not seen in B‑ALL and AML. CONCLUSION: Our study indicates that there was very good correlation of 5‑color cytoplasmic tube‑based diagnosis versus final diagnosis based on morphology, cytochemistry, and flow cytometry. We can use this 5‑color cytoplasmic tube method to make immunophenotyping cost‑effective.