Xylella fastidiosa is a plant pathogenic bacterium that causes many economically important agricultural diseases and is transmitted by the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Efficient detection of X. fastidiosa in field collected H. vitripennis in an area-wide management program can contribute to risk assessment associated with insect presence in vineyards. Prior to conducting molecular assays for detection of X. fastidiosa in individual insects, H. vitripennis must be removed from yellow sticky traps with a solvent such as orange oil. In this study, we determined the effect of orange oil concentration on extraction of individual H. vitripennis following trap removal on detection of X. fastidiosa by qRT-PCR. In a ten-fold dilution series of orange oil, increasing amounts of orange oil caused decreasing levels of X. fastidiosa detection in standardized positive samples. Additionally, tests on the effects of Stickem (R) brand trap adhesive on qRT-PCR and development of methods which lowered the concentration of orange oil often present in field samples determined the point where detection of X. fastidiosa was negatively impacted. These results benefit the monitoring and screening for Xylella fastidiosa from leafhoppers collected on sticky cards used in regulatory area-wide management.