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Human-induced changes in land and water resources adversely affect global hydrological regimes. Hydrological alteration of the natural flow regime is considered to have a significant damaging and widespread impact on river ecosystems and livelihoods. Therefore, understanding the hydrological alteration of rivers and the potential driving factors affecting such alterations are crucial to effective water resources management. This study analyses the impact of changes in land use, climate, and hydropower development on the hydrological regime of the Srepok River Basin in the Lower Mekong Region. The Lower Mekong Basin (LMB) in Southeast Asia is known for its agriculture, forests, fisheries, wildlife, and diverse natural ecosystems. Historical land use and climate change are quantified (utilising European Space Agency land cover and observed meteorological data) and correlated with the hydrological indicators using the Indicators of Hydrologic Alteration (IHA) software. Moreover, pre and post impacts on the hydrological regime by hydropower development are quantified using the Range of Variability Approach (RAV) in IHA software. The results reveal that land use, rainfall, and temperature affect different aspects of the hydrological regime, with corroborating evidence to support variation among the most correlated IHA and environmental flow component (EFC) parameters with the three drivers. The highest and lowest correlations among the IHA and EFC parameters under each driver are against land use (0.85, -0.83), rainfall (0.78, -0.54), and minimum and max temperatures (0.42, -0.47). Among the parameters, the fall rate has the most significant effect on hydrological alteration of all drivers. Hydropower development in the basin mostly affects the fall rate and reversal. Identifying the connection between these multiple drivers and hydrological alteration could help decision-makers to design more efficient and sustainable water management policies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)