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Background: Many bioinformatics pipelines are available nowadays to analyze transcriptomics data produced by high-throughput RNA sequencing. They implement different workflows that address several analysis tasks, supported by the use of third party programs. Nevertheless, a proper workflow definition for RNA-seq data analysis is still lacking. Objective: To proper define what a comprehensive RNA-seq data analysis workflow should be. Compare all available pipelines and, if such a solution is not available, implement a new pipeline. Method: We have developed a new pipeline integrating state-of-the art programs for different parts of the RNA-seq analysis. We also have used RUbioSeq libraries to achieve a scalable solution. Results: We have defined a comprehensive RNA-seq data analysis workflow, comprising the most common needs demanded by biologists and implemented it in a new pipeline, nextpresso. We also validate it in two case studies presented here. Conclusion: Nexpresso is a new, freely available, pipeline covering the most common needs of RNA-seq data analysis. It is easy to configure, generates user friendly results and scales well for larger studies comprising a high number of samples.