The accumulation of homogentisic acid (HGA) in patients with alkaptonuria is associated with the concomitant deposition of pigments contain significant amounts of polymerised HGA (polyHGA) in the bodily tissues of the patients. The polymerisation of HGA under various different conditions in vitro was investigated to attempt to understand if there is a correlation between the conditions and rate of pigment deposition in vivo. In this study, we applied a selection of different analytical chemistry techniques including: nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX), UV-visible spectroscopy (UV-visible) and electron paramagnetic resonance (EPR). We observed condition-dependent polyHGA formation, and are currently correlating these findings with literature data (e.g. histological studies) to understand polyHGA pigment deposition inside specific tissues in the body.