DEsubs is a network-based systems biology R package that extracts disease-perturbed subpathways within a pathway network as recorded by RNA-seq experiments. It contains an extensive and customized framework with a broad range of operation modes at all stages of the subpathway analysis, enabling so a case-specific approach. The operation modes include pathway network construction and processing, subpathway extraction, visualization and enrichment analysis with regard to various biological and pharmacological features. Its capabilities render DEsubs a tool-guide for both the modeler and experimentalist for the identification of more robust systems-level drug targets and biomarkers for complex diseases.