The purpose of this study was to assess the accuracy and reliability of tooth width measurements and Bolton Indices delivered by the Clin-Check Pro 6.0 software (Align Technology, San José, CA, USA). Fifty-four plaster casts were selected and measured with a manual calliper by a trained and calibrated observer. The data gathered were compared with those delivered by the software on the corresponding fifty-four virtual casts. The method reliability of the software was assessed by comparing the measurements performed by the software on 201 pairs of clin-checks corresponding to two consecutive treatment phases. Accuracy and reliability were statistically assessed using a mixed model. The software tends to provide larger widths compared with the manual method. Statistically significant differences were found in 23 teeth. At a global level, the mean difference between the methods was −0.19 mm, with a Dahlberg error of 0.24 mm and an intraclass correlation coefficient of 0.98. The Bolton Indices delivered by the two methods had a moderate correlation (ICC = 0.59; 0.69). The within method reliability of the software was extremely high. Tooth width measurements delivered by the software, despite the positive bias, can be considered accurate and clinically acceptable for all teeth except molars. The Bolton Indices made available by the software are not accurate and clinically acceptable, especially in the case of mandibular excess.