Earthworms are used as biomarkers to determine the bioavailability of contaminants. As such, their uptake of contaminants has been studied extensively. Protocols have been established to ensure that laboratory-obtained data are valid and comparable. However, the method of removing the organism’s gut content (depuration) before assessing the contaminant in the tissue is not standardized. The aim of this research project is to investigate some parameters for earthworm depuration: light conditions and coprophagy prevention. Eisenia fetida were depurated for 48 hours in two separate studies according to guidelines ASTM-E1676 and OECD Test No. 317. In one study, the subjects and material egested were assessed under conditions of continuous light and continuous darkness. In another study, three filter paper change frequencies were employed during depuration to prevent coprophagy. The earthworms depurated in continuous darkness egested 94% more material per mg of earthworm than the earthworms in continuous light. The depuration methods that included a filter paper change every 12 and 24 hours resulted in 62% and 10% more egested material per mg of earthworm than the control with no filter paper change, respectively. The results indicate that depuration would be faster and more total under continuous darkness and employing a coprophagy prevention method. These findings could lead to more efficient depuration methods.