Hydrogen peroxide (H2O2) is one of the Reactive Oxygen Species (ROS) that seems to play an essential role in pathophysiological processes. H2O2 might act as a signalling molecule and modulate different crucial cellular signalling pathways. However, identification of location where H2O2 is generated and quantification of H2O2 flux in cells and cellular compartments are virtually impossible with traditional methodology. The aim of this work was to implement and develop methodological strategies based on the use of a hydrogen peroxide biosensor, HyPer, in skeletal muscle cells, such as myoblasts, myotubes and individual muscle fibres isolated from the flexor digitorum brevis (FDB) mouse muscle. The expression of HyPer biosensor in the cytosol of these cells followed by live cell fluorescence microscopy image analysis permits to monitor and register intracellular H2O2 in situ and in real time in those cells. HyPer biosensor is synthesised intracellularly in cells. Thus, the coding DNA sequence of HyPer, inserted in a cloning vector, had to be incorporated into the cell to achieve biosensor expression. For this purpose, we carried out transfection techniques based in chemical agents for HyPer transfection in C2C12 myoblasts and C2C12 myotubes. In the case of skeletal muscle fibres, a combination of microinjection and electroporation techniques were undertaken to incorporate the coding sequence of HyPer into muscle fibres of the FDB muscle. Analysis of live cell fluorescence microscopy images confirmed both expression and functionality of HyPer as H2O2 biosensor. We monitored fluorescence emitted by cells expressing HyPer during an experimental period in which cells or isolated fibres were exposed to extracellular hydrogen peroxide in the medium or exposed to a reducing agent, dithiothreitol (DTT). An improved biosensor, HyPer2, potentially with higher H2O2 sensitivity than HyPer, was assayed in muscle fibres and proved to detect intracellular H2O2 generated by an extracellular enzymatic system, glucose oxidase (GOX) / catalase, which produces and hydrolyzes H2O2. Statistical analysis: paired Student’s t test for single comparisons and significance set at p < 0,05. Results indicate that (i) It is possible the transfection and intracellular expression of hydrogen peroxide biosensor HyPer in C2C12 myoblasts and myotubes and in single isolated skeletal muscle fibres, ii) HyPer biosensor is functional and detects changes in the intracellular concentration of hydrogen peroxide, and (iii) HyPer2 biosensor is more sensitive and detects intracellular changes of H2O2 in skeletal muscle fibres where H2O2 had been generated by an extracellular enzymatic system, GOX/catalase. In conclusion, using chemical transfection techniques and a combination of electroporation and transfection, it is possible to incorporate HyPer and HyPer2 biosensors into skeletal muscle cells and register intracellular H2O2 flow in these cells and fibres in situ and in real time.