Ilomastat is a matrix metalloproteinase inhibitor (MMPi) that has shown the potential to inhibit scarring (fibrosis) by mediating healing after injury or surgery. A long lasting ocular implantable pharmaceutical formulation of ilomastat is being developed to mediate the healing process to prevent scarring after glaucoma filtration surgery. The ilomastat implant was coated with water permeable and biocompatible phosphoryl choline polymer (PC1059) displayed extended slow release of ilomastat in vitro and in vivo. The ocular distribution of ilomastat from the implant in rabbits at day 30 post surgery was determined by the extraction of ilomastat and its internal standard marimastat from the ocular tissues, plasma, aqueous humour and vitreous fluid followed by capillary-flow liquid chromatography (cap-LC), the column effluent was directed into a triple quadrupole mass spectrometer operating in product scan mode. The lower limits of quantification (LLOQs) were 0.3 pg/μL for ocular fluids and plasma, and 3 pg/mg for ocular tissues. The extraction recoveries were 90-95% for ilomastat and its internal standard from ocular tissues. Ilomastat was found in ocular fluids and tissues at day 30 after surgery. The level of ilomastat was 18 times higher in the aqueous humour than vitreous humour. The concentration ranking of ilomastat in the ocular tissues was sclera > bleb conjunctiva > conjunctiva (rest of the eye) > cornea. Mass spectrometry analysis to confirm the presence of ilomastat in the ocular tissues and fluids at day 30 post-surgery establishes the extended release of ilomastat can be achieved in vivo, which is crucial information for optimisation of the ilomastat coated implant.