AbstractThis unit presents basic techniques for immunophenotyping by flow cytometry, direct using a conjugated monoclonal antibody and indirect using an unconjugated primary antibody followed by a conjugated secondary antibody. Combinations of these methods are described for two‐, three‐, and four‐color staining. Analysis of data acquired from cells stained by these procedures is detailed. A procedure is given for the detection of the location of nonviable cells so that they can be gated out of the analysis.Keywords: flow cytometry; immunophenotyping; indirect staining; direct staining; multicolor staining; immunophenotypic analysis.