Direct physical interaction is an essential aspect of social interaction in various mammal species. Restricting such interaction in mice lead to the observation of specific separation-induced social communication.Here, we separated sex- and age-matched mice from each other using a transparent Plexiglas divider containing a porous section at its bottom. Following this separation phase, the divider was removed, and mice were allowed to freely interact with one another. During both the separation and unification phases audio and video were recorded. Separated mice emitted a set of vocalizations distinct from previously reported ultrasonic vocalizations (USVs), referred to as separation-induced calls (SICs). Mostly conventional USVs were emitted during direct interaction. About 70% of the SICs produced had peak frequencies ranging from 6 to 46kHz (three modi at 6-8, 18-20, and 35-37 kHz), much lower than conventional USVs. Thus, SICs displayed different spectro-temporal properties compared to USVs, resulting in different SIC classes. In contrast to USVs, only about 50% of SICs occur within a bout structure. However, within bouts, inter-call-intervals were similar for SICs and USVs. Interestingly, we observed sex-specific call repertoires in both the separation and the unification phase with male mice displaying a more variable call pattern during the separation and a less variable pattern during the unification phase compared to females.Our study examines nuanced vocal communication to decipher the affective states and could thereby provide a deeper insight into the emotional state of mice.