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Methodology for extracting an effective dielectric constant of microstrip transmission lines on multilayer substrates, from measured or simulated S-parameters data, using on-chip test structures, has been demonstrated. The methodology consists of: 1) building on-chip interconnect structures usually implemented in calibration and de-embedding procedures in microwave on-wafer test and measurements – transmission lines, stubs and pad launchers; 2) extracting the effective dielectric constant from the characteristic impedance and propagation constant of these structures, fully described by the measured or EM-simulated S-parameters. The demonstrated methodology is applicable for evaluation of dielectric and semiconductor multilayer substrates, both with lossy and lossless characteristics over a broad frequency band. Another advantage is implementation of very short transmission line structures with physical dimensions much smaller than a quarter wavelength of the highest investigated band frequency, thus preserving a valuable chip area in the test structures and being compatible with some of the calibration TRL elements.