Medium chain length (C8, C10) mono-, di- and triacylglycerols (MCGs) are used in pharmaceutical formulation development of poorly soluble compounds, as a means to increase their oral bioavailability. The ratios of C8 and C10 fatty acid components along with the ratio of monoglycerides, diglycerides and triglycerides can significantly impact overall solubilizing characteristics both in vitro and in vivo. Existing literature methods either do not have the desired selectivity or simplicity to adequately characterize these MCGs. A reversed-phase HPLC method has been developed utilizing a Waters Symmetry C18 (150 mm x 4.6 mm, 3.5 microm particle size) column with a gradient of acetonitrile and water. The effluent was monitored using a UV detector at 220 nm and relative response factors were determined for all components with available standards to allow for accurate quantitation. The RP-HPLC method was optimized for selectivity, sensitivity and efficiency and was successfully applied to the characterization of commercial samples yielding superior sensitivity and ease of preparation compared to existing compendial methods.