There is no validated method of assessing masseter muscle thickness (MMT) by ultrasound imaging (US). However, this is important to ensure study and measurement quality of MMT by US in future studies, as MMT differs depending on the examined area. Thus, this study's aim was to present a new standardized method for assessing the MMT by US and to evaluate the reliability, consistency, and accuracy of its measurements. We also compared the results of MMT measurements obtained by US and computer tomography (CT). The study included nine healthy adults. The US and CT scans were collected in a supine rest position with the mandible in relaxed position. US measurements were determined according to a new standardized protocol (with precise probe location). The MMT measured by CT and US over a seven-day interval showed excellent intra-rater reliability. The mean MMT measured by CT was 12.1 mm (1.74) on the right side and 11.9 mm (1.61) on the left side. The mean MMT measured by US was 12.7 mm (2.00) on the right side and 11.5 mm (1.37) on the left side. The mean percent error in MMT measurement between CT and US was below 6%. A strong linear relationship was found between the CT and US measurements of the MMT on both body sides (p < 0.001, r ≥ 0.93). The proposed method of MMT measurement using US demonstrated excellent reliability, yielding results similar to those obtained from CT images. We recommend the use of this standardization protocol in further studies where precise assessment of MMT by US is expected.