A novel method based on Atomic Force Microscopy (AFM) and digital light microscopy (DLM) to measure the concentration of bovine insulin solution is presented. To overcome the limited sensitivities of the conventional methods of protein trace detection (such as Lowry method, Ultraviolet Absorption Method, Coomassie Bright Blue Dye-binding Method), a new approach of determining insulin concentration by calculating the volume of insulin crystals on a mica substrate using the softwares of AFM and DLM is developed. In this approach, sulfuric sodium was used as a precipitant reagent, and hydrochloric acid and sodium hydroxide were used to adjust the sample pH value to the insulin isoelectric point of ~5.35. Insulin crystals were formed by a simple evaporation method at the room temperature. The resultant crystal growth is observed and analyzed by the AFM and DLM. The up-to-date experimental results show that the combination of AFM and DLM is a feasible method to determine the concentration of insulin and other protein solutions, although the error margins must be improved in order for the method to become practical for clinical usage.