We present a novel method for analysing socio-spatial segregation in cities by considering constraints imposed by transportation networks. Using a multilayered network approach, we model the interaction probabilities of socio-economic groups with random walks and Lévy flights. This method allows for evaluation of new transport infrastructure’s impact on segregation while quantifying each network’s contribution to interaction opportunities. The proposed random walk segregation index measures the probability of individuals encountering diverse social groups based on their available means of transit via random walks. The index incorporates temporal constraints in urban mobility with a parameter, $$\alpha \in [0,1)$$
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, of the probability of the random walk continuing at each time step. By applying this to a toy model and conducting a sensitivity analysis, we explore how the index changes dependent on this temporal constraint. When the parameter equals zero, the measure simplifies to an isolation index. When the parameter approaches one it represents the city’s overall socio-economic distribution by mirroring the steady-state of the random walk process. Using Cuenca, Ecuador as a case study, we illustrate the method’s applicability in transportation planning as a valuable tool for policymakers, addressing spatial distribution of socio-economic groups and the connectivity of existing transport networks, thus promoting equitable interactions throughout the city.