A novel concept and theory of moving reaction boundary (MRB) retardation signal (RMRB) was advanced for determination of total protein content via MRB electrophoretic titration (MRBET). The theoretical results revealed that the retardation extent of boundary displacment, viz., the RMRB value, was as a function of protein content. Thus, the RMRB value of a sample could be used to determine its total protein content according to the relevant calibration curve. To demonstrate the concept and theoretical results, a novel microdevice was designed for the relevant experiments of MRBET. The microdevice has 30 identical work cells, each of which is composed of five ultrashort single microchannels (5 mm). In the microdevice, fluorescein isothiocyanate (FITC) was used to denote MRB motion and RMRB value for the first time, the polyacrylamide gel (PAG) containing protein sample was photopolymerized in microchannels, and the MRB was created with acid or alkali and target protein sample. As compared to the classic Kjeldahl method and conventional MRBET performed in glass tube, the developed titration chip has the following merits: good sensitivity (0.3-0.4 μg/mL vs 150-200 μg/mL of protein concentration, 0.6-0.8 ng vs 30-2000 μg of absolute protein content), rapid analysis (20-60 s vs 15-200 min), and portable low-power (15 V vs 200 V).