Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.