HTS remains the key methodology for finding hit compounds within the pharmaceutical industry. Large compound libraries are screened against biological targets to identify novel hit compounds. These hits are confirmed by follow up assays that yield pIC₅₀ values. Biophysical methods are rapidly gaining traction in HTS cascades to confirm target engagement prior to commencement of expensive chemistry. Differential scanning fluorimetry (DSF) is one such technique that can provide an indication of compound-target protein binding. Melting temperatures (Tm) of the target protein can be compared to the Tm measured in the presence of a potential hit compound. The difference in melting temperature (ΔTm) is concomitant with compound binding. This study evaluates the benefits of DSF as a triage assay in HTS to validate hit compounds post HTS campaigns. DSF was performed on targets from recent HTS campaigns; a kinase, GTPase and a lipase using compounds with known pIC₅₀ values. MST and SPR were performed and compared to the DSF data, to observe any agreement between these techniques. This study finds DSF an effective method to analyse protein stability with compounds in HTS. Our findings lead us to suggest the use of DSF prior to HTS campaigns, rather than as a triage activity. Consequently, the data generated from DSF could provide a strong prediction of the output quality to be expected from an HTS campaign.