(1)H-NMR spectroscopy has been used to quantify squalene in meibum and sebum. Squalene has many beneficial properties and its loss on the surface of skin upon ultraviolet light exposure or in the tear film with dry eye could be detrimental. In this study, we confirm the NMR proton resonance assignments of squalene, squalene in human meibum, and in human eyelid lipid using heteronuclear single quantum correlation spectroscopy. Our results confirm the presence of squalene in eyelid lipid. We speculate that the squalene in eyelid lipid could be secreted from sebaceous glands. The NMR resonances between 5.2 and 5.0 ppm, characteristic of the =CH of squalene, were resolved in the spectrum of human meibum and used to estimate that 1 % or less of squalene is present in meibum. However, the resonance assignments of squalene in meibum were not confirmed. The characteristics of squalene including its anti-inflammatory, antioxidant, and antibacterial qualities suggest that the presence of a squalene film is beneficial. Its loss in human meibum from patients with dry eye could be detrimental and contribute to the symptoms observed in these patients.