Research has highlighted the need for new methods to assess emotions in children on multiple levels to gain better insight into the complex processes of emotional development. The startle reflex is a unique translational tool that has been used to study physiological processes during fear and anxiety in rodents and in human participants. However, it has been challenging to implement developmentally appropriate startle experiments in children. This article describes a procedure that uses predictable and unpredictable aversive events to distinguish between phasic fear and sustained anxiety in children and adolescents. We investigated anxious responses, as measured with the startle reflex, in youths (N =36, mean age=12.63years, range=7–17) across three conditions: no aversive events (N), predictable aversive events (P), and unpredictable aversive events (U). Short-duration cues were presented several times in each condition. Aversive events were signaled by the cues in the P condition but were presented randomly in the U condition. Participants showed fear-potentiated startle to the threat cue in the P condition. Startle responses were also elevated between cues in the U condition compared with the N condition, suggesting that unpredictable aversive events can evoke a sustained state of anxiety in youths. This latter effect was influenced by sex, being greater in girls than in boys. These findings indicate the feasibility of this experimental induction of the startle reflex in response to predictable and unpredictable events in children and adolescents, enabling future research on interindividual differences in fear and anxiety and their development in youths.