Abstract Background and Aims Faecal calprotectin [FC] is a valid and non-invasive marker of mucosal inflammation. It is widely used both in clinical trials and in daily clinical practice for patients with inflammatory bowel diseases, but currently no accepted standardization for FC testing is available. Our primary aim here was to provide a clinician’s guide containing all the practical information on FC measurement in order to avoid any confounding factors, to minimize intra- and inter-individual variability in dosage, and to ensure a better and adequate interpretation of the results. Methods We conducted a detailed search of the scientific literature in the PubMed/MEDLINE, EMBASE and Cochrane databases up to January 2020 to find all relevant and available articles on pre-analytical and analytical phases of FC measurement. Results FC testing is a multi-step procedure consisting of a pre-analytical phase aimed to collect and process the stool sample and a subsequent analytical phase of FC measurement. Several factors can influence test results determining false positives or false negatives. Importantly, this faecal marker is mostly used for patient follow-up and as a predictor of treatment response. For this reason, any altered data may affect the physicians’ decisions, negatively impacting on patient management. Conclusions This review provides for the first time practical advice to minimize dosage variability, although further dedicated studies are needed to compare commercially available tests and identify the best tools for the most precise and accurate FC measurement.