Groundwater is an important component of river runoff, the quantification of which provides valuable information for water management. This study aims to evaluate the spatial and temporal variations in the contribution of groundwater to the river flow in the Mesta River Basin. Two tasks were performed with corresponding research methods and study periods. The first one aimed to determine the spatial distribution of the groundwater runoff modulus values (Mgw) by processing mean monthly flow data from numerous headwater stations for the period before the strong anthropogenic impact on the river system. The map of the groundwater runoff modulus was created based on the positive association of the Mgw values with the mean elevation of the catchments and the associated regression equation. Focusing on temporal analysis, for the second task, two-pass digital filtering was applied in order to separate groundwater from the daily time series of river runoff, which allows distinguishing the quick part of baseflow from the total groundwater runoff. The results from the application of this method showed the temporal distribution of groundwater runoff, which occurs mainly in spring, thus indicating a marked seasonality of this phenomenon. The proportion of groundwater flow in the formation of the river flow of the Mesta River Basin at Momina Kula was assessed as 26–27%. The results of the study are expected to better support water management decisions.