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Here, we present a detailed protocol for the Cell Painting PLUS (CPP) method along with an automated image and data analysis strategy. In CPP, the iterative staining, elution, and re\u2010staining of cells with seven fluorescent dyes enable multiplexed analysis of nine cellular compartments and organelles. Each dye is captured in individual imaging channels to specifically distinguish effects on the plasma membrane, actin cytoskeleton, cytoplasmic and nucleolar RNA, lysosomes, nuclear DNA, endoplasmic reticulum, mitochondria, and Golgi apparatus. During the image analysis procedure, 894 morphological features (readouts) are extracted from single cells to generate comprehensive phenotypic profiles that resemble morphological perturbations. For efficient processing of the extracted features, we provide an automated data analysis workflow, which includes quality control, data normalization, and various data visualization tools. This workflow is based on a customized\n                    <jats:italic>CPPAnalyzer<\/jats:italic>\n                    Jupyter notebook and a\n                    <jats:italic>CPPManager<\/jats:italic>\n                    KNIME workflow, which can be easily applied without any special bioinformatics knowledge. In this way, CPP expands the multiplexing capacity, customizability, and, importantly, organelle specificity of the available CP\u2010based screening methods. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Seeding of U2OS, MCF\u20107, or HepG\u20102 cells in multiwell plates and treatment of cells with compounds\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 1<\/jats:bold>\n                    : Preparation of assay\u2010ready compound plate\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Alternate Protocol<\/jats:bold>\n                    : Seeding and differentiation of primary RPTEC\u2010TERT1 cells in multiwell plates\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Staining and imaging of cells using Cell Painting PLUS\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Image analysis\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Data normalization and data quality control\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 5<\/jats:bold>\n                    : Visualization of data using\n                    <jats:italic>CPPManager<\/jats:italic>\n                    KNIME workflow\n                  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Centre for the Protection of Laboratory Animals (Bf3R) and Experimental Toxicology German Federal Institute for Risk Assessment (BfR)  Berlin Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6403-7262","authenticated-orcid":false,"given":"Jose M.","family":"Muino","sequence":"additional","affiliation":[{"name":"German Centre for the Protection of Laboratory Animals (Bf3R) and Experimental Toxicology German Federal Institute for Risk Assessment (BfR)  Berlin Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lars","family":"Valentin","sequence":"additional","affiliation":[{"name":"Innovation Centre Food Chain Modelling and Artificial Intelligence, Department Information Technology German Federal Institute for Risk Assessment (BfR)  Berlin Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2506-4297","authenticated-orcid":false,"given":"Nils","family":"K\u00f6rber","sequence":"additional","affiliation":[{"name":"Centre for Artificial Intelligence in Public Health Research Robert Koch Institute  Berlin Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3414-1640","authenticated-orcid":false,"given":"Sebastian","family":"Dunst","sequence":"additional","affiliation":[{"name":"German Centre for the Protection of Laboratory Animals (Bf3R) and Experimental Toxicology German Federal Institute for Risk Assessment (BfR)  Berlin Germany"},{"name":"Equal contribution"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2904-0271","authenticated-orcid":false,"given":"Shu","family":"Liu","sequence":"additional","affiliation":[{"name":"German Centre for the Protection of Laboratory 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