This thesis presents the development of a microfabricated high-performance liquid chromatography (HPLC) system. The design, fabrication, and characterization of individual HPLC components such as high-pressure pumps, mixers, flow sensors, composition sensors, separation columns, filters, and detectors is presented. These individual components were then integrated to create robust, feedback-driven separation systems capable of performing gradient, reverse-phase, nanoscale HPLC. Two separate separation systems were created. The first integrated system was a microfluidic device for HPLC tandem mass spectrometry (HPLC-MS/MS) designed for proteomic applications. The second system was a portable HPLC conductivity detection (HPLC-CD) system designed for point-of-care applications such as biodetection. Both systems demonstrated good performance and repeatability. The performance of these systems is largely attributable to the development of HPLC-compatible sensors that could provide precise control over the elution profiles. These microfluidic closed-loop flow control systems represent an important advancement in the microfluidics field, where open-loop flow control is universally used, and risks becoming inadequate with the increasing complexity of microfluidic systems.