The Hallmark Gene Sets were originally generated by Liberzon and colleagues to represent defined biological processes and coordinated expression pat-terns. The gene sets were identified through computational and manual curation methods to identify overlapping gene sets in Molecular Signatures Database (MSigDB) collections retaining genes that display coordinated expression. The development of this gene set helped facilitate more ro-bust Gene Set Enrichment Analysis (GSEA) results. The National Toxicology Program (NTP) toxicogenomics program uses short term exposures in rodents to assess the effects of test article exposure on gene expression. Changes in gene expression can be used to model the toxic effects of the test article. This can be done by comparing the effects with other substances in other data-bases and analysis tools. To enhance the NTP analysis of genomic effects, the Hallmark Gene Sets have been manually curated and refined for relevance to the NTP toxicogenomic study design. The curation utilized the NextBio plat-form to identify correlations between Hallmark Gene Sets and genes upregu-lated or downregulated in rodent liver. Using this information, a mechanistic interpretation was developed for the Hallmark Gene Sets that can be used to enhance GSEA for NTP studies. The Chemical Effects in Biological Systems (CEBS) database is a public resource for NTP toxicology testing data. The an-notated Hallmark Gene Set can be reviewed, searched and downloaded from the CEBS NTP Data Collections application (https://manticore.niehs.nih.gov/datasets).