For a period exceeding five decades, industrial and scientific communities, in conjunction with regulators, have utilized a complexified, standardized system (e.g., OECD, Organisation for Economic Co-operation and Development; ISO, International Organization for Standardization; ASTM, American Society for Testing and Materials; CEN, Comité Européen de Normalisation) for the estimation of biodegradability of organic compounds. This system has been adopted in numerous countries worldwide and has also been integrated into European legislation (REACH, registration, authorisation, and restriction of chemicals). In recent years, a number of deficiencies have been identified in the standardized biodegradation test systems. This comprehensive review sets out the fields in which improvements are necessary to set up the next generation of reliable, standardized biodegradation tests. The main focus of the review is the challenges and modifications needed to test difficult-to-test compounds such as volatile, hydrophobic compounds, UVCBs (unknown or variable composition, complex reaction products or biological materials), water-soluble polymers, and plastics. Recent advances in the characterization of inocula for biodegradation tests are also addressed, which offer a valuable opportunity to enhance the reliability and reproducibility of biodegradation assays. Moreover, the potential for predicting biodegradation in the environment is a subject that is discussed in this text.