This study verified the accuracy of the speed of sound (SOS) measured by the combination method, which calculates the ratio between the thickness values of cartilage measured by using the magnetic resonance imaging (MRI) and the ultrasonic pulse-echo imaging, and investigated in vivo application of this method. SOS specific to an ultrasound imaging device was used as a reference value to calculate the actual SOS from the ratio of cartilage thicknesses obtained from MR and ultrasound images. The accuracy of the thickness measurement was verified by comparing results obtained using MRI and a non-contact laser, and the accuracy of the calculated SOS was confirmed by comparing results of the pulse-echo and transmission methods in vitro. The difference between laser and MRI measurements was 0.05 ± 0.22 mm. SOS values in a human knee measured by the combination method in the medial and lateral femoral condyles were 1650 ± 79 and 1642 ± 78 m/s, respectively (p < 0.05). The results revealed the feasibility of in situ SOS measurement using the combination method.