Accurate measurement of serum gonadal steroid concentrations in children is critical for the accurate diagnosis of a multitude of pediatric endocrine disorders. Immunoassays, often "direct" without extraction or chromatographic separation have become the principal methods for determining values of testosterone and estrogen. While these assays may be validated in adults, given their higher levels of hormones, these methods do not produce accurate results in children with lower levels of these steroids. In recent years, liquid chromatography tandem mass spectrometry has emerged as the most accurate method for measuring the low androgen and estrogen levels in children. Additionally, new bioassays using recombinant engineered cells with steroid receptors hold promise for the future. Although offering a noninvasive approach, salivary testosterone measurements in children do not have proven sensitivity and specificity and cannot be generally recommended for clinical use. Clinicians must give as much attention to the methods used to determine their patients'gonadal steroid values as to the reported values themselves.