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In this paper, a simple and effective solution is proposed to accurately estimate the complex relative permittivity of individual layers and multilayers of dielectric material samples from the S-parameters measured by two waveguide cells having equal or different lengths filled with the same vacuum/empty material without having to calibrate before performing experiments. The measurement system is set up by modeling using the Computer Simulation Technology (CST) software. In the modeling, a single layer/multilayer material sample is placed in the X-band rectangular waveguide and it has two ports used for the electromagnetic wave supply and measurement of S-parameters. From the S-parameters measured, the complex relative permittivity of individual layers and the multilayers of the material samples are estimated by the proposed method. The known single-layer and multilayer materials such as Garlock, Bakelite, and Teflon have different dielectric constants and thicknesses. The results show that the complex relative permittivity of the samples matches the measured and calculated values of S-parameters in the frequency range of 8.2GHz to 12.4GHz.