The overall goal of the EnvMetaGen project No 668981 is to expand the research and innovation potential of InBIO – Research network in Biodiversity and Evolutionary Biology, through the creation of an ERA Chair in Environmental Metagenomics. This field was selected as the focus of the ERA Chair, because Environmental DNA (eDNA) analysis is increasingly being used for biodiversity assessment, diet analysis, detection of rare or invasive species, population genetics, and ecosystem functional analysis. In this context, the work plan of EnvMetaGen includes one work package (WP) dedicated to the Deployment of an eDNA Lab (WP4), which involves the training of InBIO researchers and technicians for implementing best practice protocols for the analysis of eDNA (Task 4.2). This report provides an overview of best practices for analysing eDNA samples, focusing on vertebrate faeces, water samples and bulks of invertebrates. It covers all steps from DNA extraction to amplification and sequencing in high throughput platforms, while addressing the challenges identified in each step. Moreover, it describes protocols already optimised and currently under development at InBIO, with particular reference to the detection of single species, diet assessments and biodiversity assessments. These three different outputs are fully aligned with the three key application areas that have been targeted in EnvMetaGen for a wider application of environmental metagenomics, and on which the development of strategic triple helix initiatives will focus on. These will therefore contribute to mainstream this technology as a new cost-effective solution to tackle current challenges faced by business partners and other stakeholders, while fostering InBIO-Industry-Government relationships (WP5 - Strengthening the triple helix). The protocols described herein were developed in close connection to, and build on accomplishments of, the other WPs in the project, including: i) the recruitment of the ERA Chair team (WP2), ii) secondments and researcher exchanges through collaborations with international networks (WP3), iii) the enhancement of the computational infrastructure at InBIO (WP4), and iv) participation in and organization of workshops and conferences (WP6). Future directions in eDNA analyses at InBIO, and other potential laboratory pipelines for future optimisation are identified. Together, Deliverables D4.2-D4.5 (this document; Egeter et al. 2018; Ferreira et al. 2018; Galhardo et al. 2018) form a detailed account of the successful deployment of a fully functional eDNA lab under the EnvMetaGen project, and provide a valuable resource for eDNA practitioners in all spheres of the triple-helix model.