PURPOSE.: To determine whether Nile Red and Oil Red O stains are able to detect tear film lipids deposited on silicone hydrogel contact lenses. METHODS.: Eight unworn lotrafilcon A lenses were individually soaked in successively decreasing amounts of cholesterol oleate solution (5.6 to 0.00 mg/ml) for 1 day in triplicate for each staining procedure (etafilcon A lenses were also soaked as a control). The sets of lenses were then stained with Nile Red or Oil Red O. The lenses were then individually visualized with a Nikon Eclipse 80i florescent microscope at 100× magnification, and two representative photos were taken of each lens. Both staining procedures were repeated with human worn lotrafilcon A lenses. RESULTS.: The Nile Red stain detected variable yet decreasing amounts of lipids when lenses were incubated in lipid concentrations ≥0.09 mg/ml. Oil Red O detected decreasing amounts of lipids when lenses were soaked in lipid concentrations ≥0.35 mg/ml. The Nile Red stain produced considerably more background staining than Oil Red O, and approximately half of the negative control lenses stained with Nile Red while there was minimal staining of lenses stained with Oil Red O. Etafilcon A lenses yielded decreasing amounts of lipid when soaked in successively lower concentrations of lipid when stained with Nile Red. Human-worn lotrafilcon A lenses yielded similar lipid characteristics when compared with in vitro lenses, with variable amounts of lipid detection when comparing individual subjects. CONCLUSIONS.: Nile Red and Oil Red O are both able to detect lipids on soft lenses in both in vitro and ex vivo conditions. Oil Red O appears to be a better stain for silicone hydrogel lenses as it offers a higher signal to noise ratio. Copyright © 2012 American Academy of Optometry.