Bioenergetic function is characterized with assays obtained by polarographic systems. Analog systems without data acquisition, visualization, and processing tools are used but require cumbersome operations to derive respiration rate and ADP to oxygen stoichiometry of oxidative phosphorylation (ADP/O ratio). The analog signal of a polarograhic system (YSI-5300) was digitized and a graphical user interface (GUI) was developed in MATLAB to integrate visualization, recording, calibration and processing of bioenergetic data. With the GUI, the signal is continuously visualized during the experiment and respiratory rates and ADP/O ratios can be determined. The integrated system was tested to evaluate bioenergetic function of subpopulations of mitochondria isolated from rat skeletal muscle (n = 10). Signal processing was applied to denoise data recorded at the sampling rate of 1000Hz, and maximize data decimation for computational applications. The error in calculating the bioenergetic outputs using decimated data is negligible when data are denoised. The estimate of respiration rate, ADP/O ratio and RCR obtained with denoised data at sampling rate as low as 5Hz was similar to that obtained with raw data recorded at sampling rate of 1000Hz.In summary, the integrated tools of the GUI overcome the limitations of data processing, accuracy, and utilization of analog polarographic systems.