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Skeletal remains of two equid species, Equus caballus (horse) and Equus asinus (donkey), have been found in archaeological contexts throughout Iberia since the Palaeolithic and Chalcolithic periods, respectively. These two species play different economic and cultural roles, and therefore it is important to be able to distinguish between the two species to better understand their relative importance in the past human societies. The most reliable morphological features for distinguishing between the two domesticated equids are based on cranial measurements and tooth enamel folds, leading to only a small percentage of archaeological remains that can be identified to species. Ancient DNA (aDNA) analysis can be used to reliably distinguish the two equids, but it can be cost prohibitive to apply to large assemblages, and aDNA preservation of non-cranial elements is often low. Collagen peptide mass fingerprinting by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a minimally destructive and cost-effective alternative to aDNA analysis for taxonomic determination. However, current ZooMS markers lack resolution below the genus level Equus. In this paper, we report a novel ZooMS peptide marker that reliably distinguishes between horses and donkeys using the enzyme chymotrypsin. We apply this peptide marker to taxonomically identify bones from the Iberian Peninsula ranging from the Iron Age to the Late Modern Period. The peptide biomarker has the potential to facilitate the collection of morphological data for zooarchaeological studies of equids in Iberia and throughout Eurasia and Africa. • Novel ZooMS biomarker discriminates between horses and donkeys. • Novel biomarker provided species level identification for archaeological samples identified only to Equus. • The enzyme, chymotrypsin, reliably provides unique peptide markers for ZooMS analysis. [ABSTRACT FROM AUTHOR]