Purpose. To compare various meibum collection methods and extraction techniques. Methods. Sixty subjects, all successful contact lens wearers, were seen on two visits. Meibum was collected from the lower lid of the right eye with a glass microcapillary tube, and with a Dacron swab, cytology microbrush, or spatula from the left eye. Extraction with 2:1 chloroform:methanol was done either immediately or after data collection was complete. Individual samples were divided into four equal aliquots for analysis of total lipids, cholesterol, and inorganic phosphates by assay-based techniques. Effects of collection method, extraction, and dry eye status were examined using repeated measures analysis of variance and logistic regression. Results. Total lipids showed significance for collection device (p < 0.0001) but not for extraction technique (p = 0.13) or dry eye status (p = 0.97). Dacron swab collection was associated with more total lipid on average than each other collection device (p < 0.0001). The cholesterol assay showed significance of collection device (p < 0.0001) and extraction technique (p = 0.0002) but not dry eye status (p = 0.55). Spatula collection was associated with more cholesterol on average than each other collection device (p < 0.0001). For inorganic phosphates, immediate extraction (p < 0.0001), cytology microbrush collection (p < 0.0001), and non-dry eye status (p = 0.03) were associated with the greater likelihood of detection. Conclusions. Dacron swab collection was associated with the highest average amount of total lipid detected, whereas spatula collection and immediate extraction was associated with the highest average amount of cholesterol detected. Cytology microbrush collection with immediate extraction on non-dry eye subjects was associated with the highest probability of detection of inorganic phosphates. Copyright © 2011 American Academy of Optometry.