Animated videos are one of the easiest ways to play when the child receives a medicine injection via an intravenous catheter that might reduce pain response. This study aimed to determine the effect of animation video on pain response for 3-6 years old during injection medicine via intravenous catheters. The study employed a quasi-experimental pre-test - post-test design. The intervention group (n: 107) was provided with an animation video during intravenous therapy, and the control group (n: 106) received standard care. FLACC (r: 0.845) was used to measure behavioural pain response, while heart rate was used to assess physiological pain response. Animation video with content validity index (Aiken’s V: 0.86). There were statistically significant differences in pain response, behavioral pain response, and physiological pain response between the intervention group and the control group. Animation video intervention can be used to reduce procedural pain during injection medicine intravenous therapy.