Liquid-phase exfoliation of graphene using a suitable solvent is safe, although the sonication period needs further exploration, which may affect the exfoliation process. This study investigated graphene exfoliation in chloroform through UV–Vis spectroscopy. Graphite powder at different ratios was soaked in chloroform and sonicated at different sonication times from 30 min to 180 min and then subjected to centrifugation at 4,000 rpm for 30 min. The supernatant was collected and analysed using UV–Vis spectroscopy at wavelengths between 220 nm and 800 nm. The UV absorbance intensity showed that the presence of exfoliated graphene peaks is free of interference at 120 min. A comparative study was conducted by using graphene in chloroform as controls and adding bioactive glass (BG) within graphite powder-chloroform emulsions in different concentrations at 120 min. Graphene appearance at the anticipated absorption peak at ~270 nm was observed, and BG addition led to agglomeration, which could provide an idea for a better material formulation strategy in developing films that combined graphene/ BG because of their exceptional properties suitable for diverse potential biomedical application.