Quantification of reproductive and stress hormones entirely non-invasively using amphibian urine samples has provided invaluable tool for understanding the biological functioning of reproductive and stress endocrine axis in rare, endangered and threatened species. This technical paper, I describe step by step methods for the laboratory validation of enzyme-immunoassays (EIAs) for the quantification of reproductive hormones (testosterone, oestradiol and progesterone) and stress hormone (corticosterone) entirely non-invasively in amphibian urine samples. These laboratory validation steps include (1) Accuracy/recovery checks, (2) Parallelism and (3) Quality control. I also present standard assay methods and together these will enable the advancement of non-invasive reproductive and stress endocrinology of amphibians.