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Identifies subpopulations in high-dimensional single-cell data. Cytophenograph is a computational pipeline that was developed to avoid the disadvantages of manual gating. The pipeline is developed using Python3, the clustering method adopted is a custom version of Phenograph (https://github.com/jacoblevine/PhenoGraph) where we insert a blocked seed. Besides Phenograph pipeline needs the following package installed: Pandas,Numpy,Sklearn for data parsing and exploring and openTSNE,Seaborn,Matplotlib for data visualization. This method is adaptative both in terms of dimensionality and sample size, making it suitable in a range of settings for which single-cell population structure is of interest, including other cancers or healthy tissues, and for use with other emerging single-cell technologies.