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Abstract Multiple sequence alignments and phylogenetic trees are rich in biological information and are fundamental to research in biology. PhyKIT is a tool for processing and analyzing the information content of multiple sequence alignments and phylogenetic trees. Here, we describe how to use PhyKIT for diverse analyses, including (i) constructing a phylogenomic supermatrix, (ii) detecting errors in orthology inference, (iii) quantifying biases in phylogenomic data sets, (iv) identifying radiation events or lack of resolution using gene support frequencies, and (v) conducting evolution‐based screens to facilitate gene function prediction. Several PhyKIT functions that streamline multiple sequence alignment and phylogenetic processing—such as renaming FASTA entries or tree tips—are also discussed. These protocols demonstrate how simple command‐line operations in the unified framework of PhyKIT facilitate diverse phylogenomic data analysis and processing, from supermatrix construction and diagnosis to gaining clues about gene function. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Installing PhyKIT and syntax for usage Basic Protocol 2: Constructing a phylogenomic supermatrix Basic Protocol 3: Detecting anomalies in orthology relationships Basic Protocol 4: Quantifying biases in phylogenomic data matrices and related measures Basic Protocol 5: Identifying polytomies Basic Protocol 6: Assessing gene‐gene coevolution as a genetic screen