5-HT2A agonists offer significant therapeutic potential but often produce severe off-target effects, such as cardiotoxicity from activation of the closely related 5-HT2B receptor. We demonstrate that 5-HT2A/5-HT2B receptor expression levels and the presence of receptor reserves can unmask partial agonist activity of ligands considered inactive at these receptors. Failure to account for receptor density may lead to underestimation of compound activity and off-target toxicity in pre-clinical screens, highlighting the need for diverse assay approaches in pre-clinical drug discovery to accurately predict clinical outcomes.