Ensuring onboard comfort of passengers on buses is crucial because it significantly impacts the overall quality of public transit services. Consequently, its evaluation becomes highly significant for public transport companies, offering valuable insights to facilitate continuous monitoring, assessment, and the implementation of targeted strategies to improve their services. Past research has primarily focused on separate assessments of on-board comfort, using both subjective and objective measurements. Even in cases in which both types of measurements were collected simultaneously, only one study has developed a one-size-fits-all, gradual scale for a real-time assessment. This paper builds upon this prior research by refining an existing framework, incorporating multiple comfort evaluation scales that consider objective measures, such as accelerations during horizontal curves, braking manoeuvres, and pavement irregularities. These measures are collected through intelligent transport system tools and are linked with the perceptions of different passenger segments on these measures gathered through surveys. The results were derived from a substantial dataset collected in an Italian case study and the implementation of three novel algorithms. Moreover, they contributed towards establishing multiple comfort scales within an actual operational environment. Furthermore, these findings are significant as these scales could serve as a valuable tool for regulating driver behaviour. In this way, each driver gains the ability to identify instances of passenger discomfort in real time and implement corrective actions to enhance overall comfort.