Threatened species are susceptible to irreversible population decline caused by adverse sub-lethal effects of chemical contaminant exposure. It is therefore vital to develop the necessary tools to predict and detect these effects as early as possible. Biomarkers of contaminant exposure and effect are widely applied to this end, and a significant amount of research has focused on development and validation of sensitive and diagnostic biomarkers. However, progress in the use biomarkers that can be measured using non-destructive techniques has been relatively slow and there are still many difficulties to overcome in the development of sound methods. This paper systematically quantifies and reviews studies that have aimed to develop or validate non-destructive biomarkers in wildlife, and provides an analysis of the successes of these methods based on the invasiveness of the methods, the potential for universal application, cost, and the potential for new biomarker discovery. These data are then used to infer what methods and approaches appear the most effective for successful development of non-destructive biomarkers of contaminant exposure in wildlife. This review highlights that research on non-destructive biomarkers in wildlife is severely lacking, and suggests further exploration of in vitro methods in future studies.