======================== Purpose of this document ======================== Environmental DNA (eDNA) underlies non-invasive, reliable, and cost-effective molecular approaches that are increasingly being used for species monitoring, biodiversity assessment, and tracking pathogens. eDNA refers to genetic material sampled from environmental sources such as water, soil, or air, without directly sampling tissue. eDNA derives from various sources, including intact cells, cellular organelles (mitochondria and chloroplasts), DNA adsorbed onto particles, pieces of tissue, or even whole microorganisms. eDNA sampling typically involves collecting environmental samples and isolating DNA, often through filtration, centrifugation, or precipitation. These approaches rely on the fact that organisms leave their DNA in the environment, making it possible to know which species are present without seeing or capturing them. However, reliable eDNA data depend on rigorous field and lab technique, and properly trained personnel because significant biases can occur throughout the process from sample collection to data processing and interpretation; such biases can greatly influence species detection results. We began writing this document as an accompaniment for an eDNA workshop offered at our Queen’s University Biological Station (`https://qubs.ca `_). The overarching goal of the workshop and thus this manual is to provide a general overview of the procedures used in eDNA studies for both single-species and multi-species detections. This 5-day workshop was designed to benefit those interested in undertaking eDNA studies themselves, or in reading and interpreting the peer-reviewed literature, or in evaluating proposals, especially for management and monitoring applications. The workshop includes lectures, demonstrations and practical training but does not require detailed prior knowledge of genetics (we do though assume that participants know the basics of DNA and simple lab methods). In the workshop and in this manual too, we offer a background on eDNA origins, methods, and applications with key emphases on how to design, implement, analyse, and interpret data from species-specific and multi-species eDNA methods. Advantages and limitations of each approach are provided, as well as the precautions needed through the whole workflow to obtain robust data. In the workshop, basic theory is be complemented by hands-on training in field and lab protocols. This document presents an overview of eDNA approaches and provides guidance from study design, sampling, sample processing and storage, laboratory methods, data processing to interpretations. It complements the theoretical and hands-on training that is provided during the workshop. We provide a glossary at the end of the document.