Flow cytometry has emerged as an important technology for immunophenotyping of acute leukemias. This chapter describes in detail, the protocol for sample preparation for flow cytometry, application of flow cytometry in diagnosis of AML, ALL (B and T) and biphenotypic leukemia. It touches upon the role of flow cytometry in minimal residual disease detection, body fluid analysis, FNAC and discusses the differences between flow cytometry and immunohistochemistry. It also elaborates upon the trouble shooting in flow cytometry.