Background & Aims: The potential of mechanistic biomarkers to improve prediction of drug-induced liver injury (DILI) and hepatic regeneration is widely acknowledged. We sought to determine reference intervals for new biomarkers of DILI and regeneration, as well as to characterize their natural variability and impact of diurnal variation. Methods: Serum samples from 227 healthy volunteers were recruited as part of a cross-sectional study; of these, 25 subjects had weekly serial sampling over 3 weeks, while 23 had intensive blood sampling over a 24h period. Alanine aminotransferase (ALT), MicroRNA-122 (miR-122), High Mobility Group Box-1 (HMGB1), total Keratin-18 (K18), caspase-cleaved Keratin-18 (ccK18), Glutamate Dehydrogenase (GLDH) and Macrophage Colony-Stimulating Factor-1 (CSF-1) were assayed. Results: Reference intervals were established for each biomarker based on the 97.5% quantile (90% CI) following the assessment of fixed effects in univariate and multivariable models. Intra-individual variability was found to be non-significant, and there was no significant impact of diurnal variation. Conclusions: Reference intervals for novel DILI biomarkers have been described. An upper limit of a reference range might represent the most appropriate mechanism to utilize these data. These data can now be used to interpret data from exploratory clinical DILI studies and to assist their further qualification as required by regulatory authorities.