MOTIVATION: The forskolin-induced swelling (FIS) assay has become the preferential assay to predict the efficacy of approved and investigational CFTR-modulating drugs for individuals with cystic fibrosis (CF). Currently, no standardized quantification method of FIS data exists thereby hampering inter-laboratory reproducibility. RESULTS: We developed a complete open-source workflow for standardized high-content analysis of CFTR function measurements in intestinal organoids using raw microscopy images as input. The workflow includes tools for (i) file and metadata handling; (ii) image quantification and (iii) statistical analysis. Our workflow reproduced results generated by published proprietary analysis protocols and enables standardized CFTR function measurements in CF organoids. AVAILABILITY AND IMPLEMENTATION: All workflow components are open-source and freely available: the htmrenamer R package for file handling https://github.com/hmbotelho/htmrenamer; CellProfiler and ImageJ analysis scripts/pipelines https://github.com/hmbotelho/FIS_image_analysis; the Organoid Analyst application for statistical analysis https://github.com/hmbotelho/organoid_analyst; detailed usage instructions and a demonstration dataset https://github.com/hmbotelho/FIS_analysis. Distributed under GPL v3.0. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.