Meibomian gland dysfunction (MGD) produces alterations in the tear film (TF) lipid layer that causes excessive evaporation of tears from the ocular surface and subsequent increased osmolarity leading to dry eye disease. However, the molecular mechanism underlying TF evaporation has yet to be understood. This study aimed to investigate the association between (o-acyl)-omega-hydroxy fatty acids (OAHFAs) derived from TF and meibum and TF evaporation in a cohort of healthy and MGD subjects.