Introduction Many patients referred for suspicion of myelodysplastic neoplasm (MDS) are subjected to unnecessary discomfort from bone marrow aspiration, due to the low disease prevalence in this population. Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression could rule out MDS with sensitivity and negative predictive value estimates close to 100%, ultimately obviating the need for bone marrow aspiration in up to 35% of patients. However, the generalisability of these findings is uncertain due to the limited sample size, the enrolment of patients at a single study site, and the reliability issues associated with laboratory-developed tests and varying levels of operator experience. This study aims to validate the accuracy attributes of peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis in an independent multicentre sample. Methods and analysis The MPO-MDS-Valid project is a cross-sectional diagnostic accuracy study comparing an index test to a reference standard. Consecutive adult patients referred for suspicion of MDS are being recruited at seven university hospitals and one cancer centre in France. At each site, flow cytometric analysis of peripheral blood samples is performed by operators who are blinded to the reference diagnosis. A central adjudication committee whose members are unaware of the index test results will determine the reference diagnosis of MDS, based on cytomorphological evaluation of bone marrow performed in duplicate by experienced hematopathologists. The target sample size is 400 patients and the anticipated study recruitment completion date is 31 December 2025. Ethics and dissemination An institutional review board (Comité de Protection des Personnes Nord-Ouest III, Caen, France) approved the protocol, prior to the start of the study. Participants are recruited using an opt-out approach. Efforts will be made to publish the primary results within 6 months after study completion. Trial registration number NCT05175469 .