A great deal of effort is being dedicated to the development of new devices able to conduct effective in vitro toxicology analyses. This paper describes the use of a microfluidic gradient maker for the toxicological analysis of two conventional local anesthetics, bupivacaine and lidocaine on cell cultures. The microfluidic device was designed and simulated using COMSOL Multiphysics and the concentration gradient in the microfluidic network was analysed through a fluidodynamic and diffusive study. Subsequently the device was fabricated with soft lithography, casting PDMS in a master to obtain channels about 250 microm deep. Both drugs were tested on C2C12 myoblasts and an analysis was performed using propidium iodide staining followed by an imaging processing routine to obtain quantitative dose-response profiles in the gradient maker. The system was critically compared with microwell-based toxicity testing. The results show that the GM is a more sensitive method for detection of cell toxicity, and compared with testing of drug toxicity using microwells with individual cell cultures, allows one shot testing with a single cell culture exposed to a large number of concentrations. However, the flow rates required to obtain a suitable concentration range across the device may damage shear sensitive cells.