Acidosis is a hallmark of most pathological conditions due to the near ubiquitous involvement of inflammation, ischaemia and cell lysis (most often in combination) and is becoming recognised as a damage associated molecular pattern in its own right [1]. Acid-sensing ion channels (ASICs) are widely expressed in neuronal and non-neuronal cells and are emerging as key pattern recognition receptors and promising drug targets for acidosis-mediated inflammation, pain and cell damage. Hi1a is a 76 amino acid, double inhibitor cystine knot venom peptide that is the most potent known inhibitor of acid-sensing ion channel 1a (ASIC1a) and has shown promising therapeutic potential in animal models of ischaemic stroke [2] and ischaemic heart damage [3]. We carried out mutagenesis studies on the peptide and channel to understand the interaction of Hi1a with ASIC1a and found: the peptide has an extensive interaction surface on the ion channel, consistent with its high avidity, and that the linker connecting the two ICK domains is crucial for its mechanism of action. We have also labelled the peptide with fluorescent or radioactive probes to facilitate in vivo biodistribution and target engagement studies in naïve mice compared to a mouse model of multiple sclerosis (experimental autoimmune encephalitis- EAE). Peripheral administration of Hi1a (IV injection) and monitoring biodistribution via dynamic PET/CT showed that although the peptide appears to clear from the plasma within 20 minutes, a substantial fraction remains in circulation for longer than 90 mins. We also show that following IV injection, fluorescently labelled Hi1a accumulates in the spinal cord of EAE mice but not in those of naïve mice, consistent with a potent therapeutic effect in this mouse model. These advances in our understanding the structure-activity relationship, biodistribution and target engagement of Hi1a in preclinical mouse models of disease should help guide studies on this promising peptide through further preclinical development.