In the realm of new economic geography studies, an ongoing debate centers around the role of transportation costs in redirecting economic activity away from peripheral areas towards urban spaces. This theory gains support from the phenomenon of rural depopulation in developed countries, as it is generally accompanied by improvements in accessibility due to new infrastructure and technology. This study introduces a methodology for analyzing the relationship between accessibility and regional inequality while accounting for rural depopulation. Moreover, it acknowledges the significance of the measurement timeframe, as the dynamics between accessibility components are not always synchronous. The developed methodology is employed for investigation within the geographical region of main-land Greece. The findings reveal that relative accessibility, in contrast to absolute accessibility, exhibits a correlation with rural depopulation in mainland Greece. Finally, the study examines the Modified Areal Unit Problem (MAUP) to address inconsistencies observed in similar spatial studies. The disparities in results among the various geographical administrative levels carry substantial implications for policymakers dealing with issues of urbanization and uneven regional development.