Investigating the growth behavior of plant root systems as a function of soil water is considered an important information for the study of root physiology. A non-invasive tool based on electromagnetic wave transmittance in the microwave frequency range, operating close to 4.8 GHz, was developed using microstrip patch antennas to determine the volumetric moisture of soil in rhizoboxes. Antennas were placed on both sides of the rhizobox and, using a vector network analyzer, measured the S parameters. The dispersion parameter S21 (dB) was also used to show the effect of different soil types and temperature on the measurement. In addition, system sensitivity, reproducibility and repeatability were evaluated. The quantitative results of the soil moisture, measured in rhizoboxes, presented in this paper, demonstrate that the microwave technique using microstrip patch antennas is a reliable, non-invasive and accurate system, and has shown potentially promising applications for measurement of rhizobox-based root phenotyping.