Abstract Summary bollito is an automated, flexible and parallelizable computational pipeline for the comprehensive analysis of single-cell RNA-seq data. Starting from FASTQ files or preprocessed expression matrices, bollito performs both basic and advanced tasks in single-cell analysis integrating >30 state-of-the-art tools. This includes quality control, read alignment, dimensionality reduction, clustering, cell-marker detection, differential expression, functional analysis, trajectory inference and RNA velocity. bollito is built using the Snakemake workflow management system, which easily connects each execution step and facilitates the reproducibility of results. bollito’s modular design makes it easy to incorporate other packages into the pipeline enabling its expansion with new functionalities. Availability and implementation Source code is freely available at https://gitlab.com/bu_cnio/bollito under the MIT license. Supplementary information Supplementary data are available at Bioinformatics online.