Neurofilament proteins have been validated as specific body fluid biomarkers of neuro-axonal injury. The advent of highly sensitive analytical platforms that enable reliable quantification of neurofilaments in blood samples and simplify longitudinal follow-up has paved the way for the development of neurofilaments as a biomarker in clinical practice. Potential applications include assessment of disease activity, monitoring of treatment responses, and determining prognosis in many acute and chronic neurological disorders as well as their use as an outcome measure in trials of novel therapies. Progress has now moved the measurement of neurofilaments to the doorstep of routine clinical practice for the evaluation of individuals. In this Review, we first outline current knowledge on the structure and function of neurofilaments. We then discuss analytical and statistical approaches and challenges in determining neurofilament levels in different clinical contexts and assess the implications of neurofilament light chain (NfL) levels in normal ageing and the confounding factors that need to be considered when interpreting NfL measures. In addition, we summarize the current value and potential clinical applications of neurofilaments as a biomarker of neuro-axonal damage in a range of neurological disorders, including multiple sclerosis, Alzheimer disease, frontotemporal dementia, amyotrophic lateral sclerosis, stroke and cerebrovascular disease, traumatic brain injury, and Parkinson disease. We also consider the steps needed to complete the translation of neurofilaments from the laboratory to the management of neurological diseases in clinical practice. Neurofilaments have been validated as specific body fluid biomarkers of neuro-axonal injury. In this Review, Khalil and colleagues provide an update on the structure and function of neurofilaments, analytical approaches and challenges in different clinical contexts, and progress towards clinical application of neurofilaments as a biomarker in various neurological disorders. Key points: Neurofilament proteins have emerged as one of the most important body fluid biomarkers of neuro-axonal injury in a wide range of neurological diseases. High-sensitivity analytical platforms enable reliable quantification of neurofilament light chain (NfL) levels in blood samples, paving the way for their use in clinical practice. Establishment of large reference databases of physiological blood levels of NfL adjusted for age and BMI was a major milestone towards the clinical use of NfL. Neurofilament levels can often not be used to diagnose disease entities but are useful as a diagnostic type biomarker in the preclinical phases of neurodegenerative diseases and as markers of disease progression, prognosis, and treatment response. Neurofilament levels are increasingly used as an outcome measure in clinical trials; FDA approval of tofersen was based on changes in blood NfL levels, marking a paradigm shift in the importance of biomarkers in regulatory approvals. Standardization and cross-compatibility of neurofilament measures taken with current emerging analytic platforms are key to completing the translation of neurofilaments into clinical practice. [ABSTRACT FROM AUTHOR]