MetaboAnalyst 4.0 is a comprehensive web-based tool for metabolomic data analysis, interpretation, and integration with other omics data. Since its last major update in 2015, MetaboAnalyst has evolved based on user feedback and technological advancements. Version 4.0 introduces four new key features: (1) real-time R command tracking and display with a companion MetaboAnalystR package; (2) a MS Peaks to Pathways module for predicting pathway activity from untargeted mass spectral data using the mummichog algorithm; (3) a Biomarker Meta-analysis module for robust biomarker identification through multiple datasets; and (4) a Network Explorer module for integrative analysis of metabolomics, metagenomics, and transcriptomics data. The user interface has been reengineered for a modern look and feel, and the underlying knowledgebases have been updated with the latest data from the Human Metabolome Database (HMDB). A Docker image is available for easy download and local installation. MetaboAnalyst 4.0 is freely available at http://metaboanalyst.ca. The user interface has been upgraded to provide a more modern look and feel while maintaining an easy-to-use modular analytical pipeline. The 12 modules are organized into four categories: exploratory statistical analysis, functional analysis, data integration and systems biology, and data processing & utility functions. The functional analysis category now includes a new module for pathway activity prediction from MS data. The data integration and systems biology category includes three modules: biomarker meta-analysis, joint pathway analysis, and network explorer. The data processing and utility functions category contains common data processing tools and links to web-based tools for NMR, GC-MS, and LC-MS spectral analysis. The MetaboAnalystR package allows users to 'see' and save the R code that MetaboAnalyst is running in real-time, which can be used locally to reproduce the analytical workflow. This improves transparency and reproducibility. The knowledgebase has been updated with HMDB Version 4.0, including updates to HMDB identifiers and links to other databases. The compound database has been expanded to include ~19,000 compounds, representing the core subset of HMDB compounds with more detailed annotations. The MS Peaks to Pathways module uses the mummichog algorithm to predict pathway activities from high-resolution MS peaks without performing accurate peak annotation. The Biomarker Meta-analysis module supports the identification of robust biomarkers through meta-analysis of multiple datasets. The Network Explorer module supports integrative analysis of metabolomics, metagenomics, and transcriptomics data through knowledge-based network analysis and visualization. Other updates include a Docker image for easy download and local installation, and links to web-based tools for spectral analysis. MetaboAnalyst 4.0 offers comprehensive support for statistical analysis, functional interpretation, and integration with other omics data. It supports real-time interactive dataMetaboAnalyst 4.0 is a comprehensive web-based tool for metabolomic data analysis, interpretation, and integration with other omics data. Since its last major update in 2015, MetaboAnalyst has evolved based on user feedback and technological advancements. Version 4.0 introduces four new key features: (1) real-time R command tracking and display with a companion MetaboAnalystR package; (2) a MS Peaks to Pathways module for predicting pathway activity from untargeted mass spectral data using the mummichog algorithm; (3) a Biomarker Meta-analysis module for robust biomarker identification through multiple datasets; and (4) a Network Explorer module for integrative analysis of metabolomics, metagenomics, and transcriptomics data. The user interface has been reengineered for a modern look and feel, and the underlying knowledgebases have been updated with the latest data from the Human Metabolome Database (HMDB). A Docker image is available for easy download and local installation. MetaboAnalyst 4.0 is freely available at http://metaboanalyst.ca. The user interface has been upgraded to provide a more modern look and feel while maintaining an easy-to-use modular analytical pipeline. The 12 modules are organized into four categories: exploratory statistical analysis, functional analysis, data integration and systems biology, and data processing & utility functions. The functional analysis category now includes a new module for pathway activity prediction from MS data. The data integration and systems biology category includes three modules: biomarker meta-analysis, joint pathway analysis, and network explorer. The data processing and utility functions category contains common data processing tools and links to web-based tools for NMR, GC-MS, and LC-MS spectral analysis. The MetaboAnalystR package allows users to 'see' and save the R code that MetaboAnalyst is running in real-time, which can be used locally to reproduce the analytical workflow. This improves transparency and reproducibility. The knowledgebase has been updated with HMDB Version 4.0, including updates to HMDB identifiers and links to other databases. The compound database has been expanded to include ~19,000 compounds, representing the core subset of HMDB compounds with more detailed annotations. The MS Peaks to Pathways module uses the mummichog algorithm to predict pathway activities from high-resolution MS peaks without performing accurate peak annotation. The Biomarker Meta-analysis module supports the identification of robust biomarkers through meta-analysis of multiple datasets. The Network Explorer module supports integrative analysis of metabolomics, metagenomics, and transcriptomics data through knowledge-based network analysis and visualization. Other updates include a Docker image for easy download and local installation, and links to web-based tools for spectral analysis. MetaboAnalyst 4.0 offers comprehensive support for statistical analysis, functional interpretation, and integration with other omics data. It supports real-time interactive data