In order to support drug discovery and development studies within the pharmaceutical industry there has been an increased use of innovative bioanalytical assays and associated analytical technology. Performing quantitative bioanalysis in a variety of biological matrices can also involve the use of sample preparation techniques, complex HLPC column switching and microfluidic systems. Development of assays for diverse therapeutic agents in biomatrices, such as plasma and urine, can be very technically challenging to obtain the sensitivity, speed and specificity required. This challenge focuses on the quantification of drugs and metabolites at very low concentration levels, in an excess of biological matrix and in a high-throughput manner. One area of wide interest is the use and application of monolithic phases where emerging technology has been implemented successfully. This review presents an overview of the application of monolithic phases in a bioanalytical setting, including the bioanalytical challenges that need to be overcome; the synthesis, use and applicability of monolithic phases (with emphasis on polymer-based phases); the currently available bioanalytical techniques and approaches; and future possibilities for these phases.