Meibomian Glands (MG) are sebaceous glands responsible for the production of meibum, the main component of the Tear Film Lipid Layer (TFLL). The TFLL facilitates the spread of the tear film over the ocular surface, provides stability and reduces tear evaporation. Alterations in meibum composition lead to different ocular alterations like Meibomian Gland Dysfunction (MGD) and subsequent Evaporative Dry Eye (EDE). The aim of the present study was to investigate the composition and abundance of meibum lipids and their relationship with eyelid margin abnormalities, lipid layer patterns and MG status. The study utilizes a lipidomic approach to identify and quantify lipids in meibum samples using an Elute UHPLC system. This system considered all four dimensions (mass/charge, retention time, ion mobility and intensity) to provide the accurate identification of lipid species. Samples were categorized as healthy or low/no signs of alteration (group 1) or severe signs of alteration or EDE/MGD (group 2). The current investigation found differences in Variable Importance in Projection lipid abundance between both groups for the MGD signs studied. Changes in meibum composition occur and are related to higher scores in eyelid margin hyperaemia, eyelid margin irregularity, MG orifice plugging, MG loss and lipid layer pattern.