The segregation is a complex phenomenon driven by the demographic composition of the population, socioeconomic factors and underlying configuration of the geographic space. Here, we focused on segregation over 24 hours that occurs during one day inferred through the mobility of people. We utilized mobile phone data from telecom provider to estimate population mobility and explore patterns of segregation in the city. We used city’s districts as spatial units for measuring segregation and quantified mobility between districts from call detail records data for the municipality of Novi Sad provided by Telecom Serbia. Following indices were calculated: Gini, Information Theory, Moran and Duncan Dissimilarity Index. Obtained results unveiled when and where the segregation is low and when and where it reaches high values. This served us to classify the districts based on their segregation level.