The occurrence of phase II metabolism via conjugation pathway, has been observed for not only several endogenous molecules such as bilirubin, bile acids, steroid hormones (androgens and estrogens), retinoids, thyroid hormones, leukotriene B4, hydroxy metabolites of arachidonic and linoleic acids, several phenolic neurotransmitters etc. but also for a number of xenobiotics belonging to diversified chemical classes. Although topic of focus in this review is pertaining to glucuronidation, other conjugation phase II reactions are known to occur via sulfation, acetylation, methylation or simply via direct addition of glycine or glutathione adducts. The review covers the importance of glucuronidation while accounting for various types of known glucuronidation reactions. The formation of acyl glucuronides and the liability of acyl glucuronides to form chemically reactive species via acyl migration mechanism are also covered in this review. A concise review of the literature on bioanalytical aspects of the analysis of glucuronides revealed that application of HPLC with UV, fluorescence, coulometric or mass spectral detections have been generally practiced for the determination of several types of glucuronides in biological fluids. Several examples are provided in this review for the estimation of glucuronides under various sub-heading such as direct estimation of nonchiral glucuronides, direct estimation of diastereomeric glucuronides, estimation followed by derivatization, estimation by using ion-pairing agent, estimation of mixed glucuronides by HPLC and/or LC-MS/MS. Additionally, several aspects pertaining to the development of sound bioanalytical methodology including aspects for handling procedures and a validation framework for establishing glucuronide assay are presented.