Therapeutic drug monitoring (TOM) focuses on measurement of drugs with narrow therapeutic windows. Inappropriate concentrations of these drugs can elicit either an absence of therapeutic response or toxic effects. Microneedles (MN) have previously been studied for their ability to painlessly penetrate the stratum corneum, allowing transdermal delivery of various compounds. Interest in MN technology to sample analytes is increasing due to their minimally-invasive characteristics when compared to conventional needles. . . To overcome problems associated with the use of conventional needles, this study aimed to identify the most suitable polymeric MN array design and utilise this, for the first time, as a sampling device capable of imbibing large amounts of fluid for TOM. Hydrogel-forming MN arrays were fabricated from blends of poly(methyl vinyl ether-eo-maleic acid) crosslinked with poly(ethylene glycol) both with and without pore-forming agents. These MN were used in combination with reverse iontophoresis and hygroscopic Iyophilised tablets to further enhance fluid uptake by polymeric matrices. Theophylline, a bronchodilator with a narrow therapeutic window, was chosen as the model drug of the study. A high-performance liquid chromatography (HPLC) method was developed and validated for detection of theophylline. This study proved the capacity of hydrogel-forming MN to take up theophylline, in vitro and in vivo, and release it in water solution for HPLC quantification. Theophylline was successfully detected and quantified from 12 of 24 MN after 1 h insertion in the back of rats dosed with 10 mg theophylline/kg body mass. While this study posed certain challenges, including problems optimising HPLC methodology and difficulties designing a method to extract theophylline in vivo, the potential of MN as a novel in vivo TOM technology with significant clinical potential was proven. Ideally, given the findings of this study, future work will focus on the use of hydrogel-forming MN for TOM in human volunteers.