For parenteral biopharmaceuticals, subcutaneous diffusion and, in the case of solid implants or suspensions, dissolution may govern the clinical profile of the drug product. Insight into the dissolution and diffusion processes of biopharmaceuticals after parenteral administration is fundamental in the development of new protein drug formulations. Using insulin as a model compound, the aim of this work was to develop a UV imaging-based method to study the real-time dissolution and diffusion behavior of solid protein drugs under stagnant conditions in a hydrogel matrix mimicking the subcutaneous tissue. Dissolution of proteins and peptides is a complex phenomenon as it may be coupled to the complicated acid base properties of these substances. UV imaging allowed the real-time dissolution and diffusion processes of insulin at different pH values and of different insulins to be studied. Dissolution rates were obtained, and the quantitative performance of the developed UV imaging method was verified. It was shown that the UV imaging dissolution method was able to differentiate between the behavior of different insulins and that human insulin dissolution was highly dependent on pH. pH effects in the microenvironment of the human insulin compacts at pH 7.40 and 3.00 were observed by UV–Vis imaging, explaining the different dissolution kinetics of human insulin at pH 7.40 and 3.00 as compared to pH 5.40. In conclusion, UV–Vis imaging may be a useful tool for studying dissolution, diffusion and pH effects in the vicinity of solid protein drug in a hydrogel matrix with the aim of achieving a better understanding of in vivo dissolution processes.