Pairwise similarity mapping (PaSiMap), implemented in Jalview, serves as an alternative to principal component analysis (PCA) for analyzing protein sequence relationships without requiring pre-computed multiple sequence alignments. It excels at distinguishing between systematic and random differences in datasets. Here, we present a protocol for sequence clustering with PaSiMap in Jalview. We describe steps for installation, sequence importing, and running PaSiMap analyses. We also detail procedures for plotting and data interpretation in RStudio, offering a streamlined approach for visualizing and analyzing protein sequence relationships effectively.