Place it at the end of a short refusal scene, right after the question appears on screen. Follow it with the Anime Ahh for a surprised reaction, then the Awkward Moment Sound Effect to hold the beat after the no before the next cut.
About this sound
This three-second voice clip catches a small character saying “iya,” Japanese for no or I don’t want to, in a soft, high-pitched tone. It feels shy and stubborn rather than angry, like a childlike protest that is more endearing than dramatic. It comes from Chiikawa, the Japanese manga and anime about tiny cute characters in everyday comic situations.