The multimodal and dispersive character of ultrasonic guided waves (UGW) offers the potential for non-destructive evaluation of fiber-reinforced composite (FRC) materials. In this study, a methodology for in situ stiffness assessment of FRCs using UGWs is introduced. The proposed methodology involves a comparison between measured wave speeds of the fundamental symmetric and antisymmetric guided wave modes with a pre-established dataset of UGW speeds and translation of them to corresponding stiffness properties, i.e., 𝐴𝐵𝐷-components, in an inverse manner. The dispersion relations of guided waves have been calculated using the semi-analytical finite element method. First, the performance of the proposed methodology has been assessed numerically. It has been demonstrated that each of the independent 𝐴𝐵𝐷-components of the considered laminate can be approximated with an error lower than 10.4% compared to its actual value. The extensional and bending stiffness properties can be approximated within an average error of 3.6% and 9.0%, respectively. Secondly, the performance of the proposed methodology has been assessed experimentally. This experimental assessment has been performed on a glass fiber-reinforced composite plate and the results were compared to mechanical tensile and four-point bending tests on coupons cut from the plate. Larger differences between the estimated 𝐴𝐵𝐷-components according to UGW and mechanical testing were observed. These differences were partly attributed to the variation in material properties across the test plate and the averaging of properties over the measurement area.