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As part of the Deepwater Horizon toxicity testing program, a number of laboratories generated oil slicks in the laboratory to study potential toxic effects of these oil slicks on aquatic organisms. Understanding the details of how these slicks affect aquatic organisms requires careful correlation between slick thickness and the observed detrimental effects. Estimating oil film thickness on water can be challenging since the traditional color-based technique used in the field is very imprecise. Also, as we demonstrate here, the films formed on the water surface are highly nonuniform on a microscale level, and thus uniform thin film thickness measurement techniques based on optical interference do not work. In this paper, we present a method that estimates the local thickness of weathered oil slicks formed on artificial seawater using light transmission and Beer–Lambert’s law. Here, we demonstrate results of careful calibration together with the actual thickness estimation. Due to the heterogeneity of the slicks formed, we present slick thickness as a range of thicknesses collected from multiple points within the oil slick. In all the experiments we used oil samples provided by the Natural Resource Damage Assessment toxicity testing program for the Deepwater Horizon oil spill. Therefore, this study has an important practical value and successfully addresses unique challenges related to measurements involving complex, viscous, paste-like heterogeneous substances such as weathered crude oil.