This study aimed to evaluate the correlation between motor lateralisation, temperament, and serum levels of cortisol. The study group consisted of 39 clinically healthy dogs: 21 females and 18 males. Lateralisation and temperament were assessed during periodic animal health checks using a standardised tape test and a proprietary assessment algorithm. The median cortisol level in the dogs was 4.6 ng/ml. The median cortisol level was 4.31 ng/ml in dogs under 3 years of age and 4.92 ng/ml in dogs over 3 years of age. The median cortisol level was 4.21 ng/ml in left-pawed dogs and 4.8 ng/ml in right-pawed dogs. Statistically significant differences were found between cortisol levels and temperament. The median cortisol level was 5.06 ng/ml in extrovert dogs and 4 ng/ml in introvert dogs.