Background: Avoidance of colonoscopy is often associated with fear, embarrassment, and disgust aroused in screening procedures. However, each emotion can be linked to different challenges faced by patients. More research is needed to evaluate and address the causes of these respective emotions. Aim: The purpose of this study was to develop and assess the scales of three negative emotions (i.e., fear, embarrassment, and disgust) caused by specific issues in colonoscopy screening. Method: The measurement items were developed based on multiple common barriers in colonoscopy screening procedures. An online sample of 232 adults aged 45-75 was recruited from Amazon Mechanical Turk to test the scales. Explorative and confirmatory factor analyses were conducted to validate the measurement models. Results: Psychometric evidence demonstrated the factor structures of three negative emotions. Each emotional factor was caused by unique combinations of barriers in the preparation, screening, and recovery stages of colonoscopy. Most of the emotional factors were associated with attitudes and screening intention. Conclusion: This study showed different dimensions of negative emotions and their underlying causes in colonoscopy. These findings will help assess specific causes of negative emotions in colonoscopy and develop effective interventions to improve screening uptake.