Endocrine-disrupting chemicals (EDCs) are xenobiotics presented in a variety of everyday products that may disrupt the normal activity of hormones. Exposure to bisphenol A as EDC at trace and ultra-trace levels is associated with adverse health effects, and children are recognized as the most vulnerable group to EDCs exposure. In this review, a summary is presented of up-to-date sample preparation methods and instrumental techniques applied for the detection and quantification of bisphenol A and its structural analogues in various biological matrices. Biological matrices such as blood, cell-free blood products, urine, saliva, breast milk, cordial blood, amniotic and semen fluids, as well as sweat and hair, are very complex; therefore, the detection and later quantification of bisphenols at low levels present a real analytical challenge. The most popular analytical approaches include gas and liquid chromatography coupled with mass spectrometry, and their enhanced reliability and sensitivity finally allow the separation and detection of bisphenols in biological samples, even as ultra-traces. Liquid/liquid extraction (LLE) and solid-phase extraction (SPE) are still the most common methods for their extraction from biological matrices. However, many modern and environmentally safe microextraction techniques are currently under development. The complexity of biological matrices and low concentrations of analytes are the main issues for the limited identification, as well as understanding the adverse health effects caused by chronical and ubiquitous exposure to bisphenols and its analogues.