The aim was to validate a newly developed methodology of semi-automatic image analysis to analyze microglial morphology as marker for microglial activation in ionized calcium-binding adaptor protein-1 (IBA-1) stained brain sections. The novel method was compared to currently used analysis methods, visual characterization of activation stage and optical density measurement, in brain sections of young and aged rats that had undergone surgery or remained naοve. The cell body to cell size ratio of microglia was strongly correlated to the visual characterization activation stage. In addition, we observed specific surgery and age-related changes in cell body size, size of the dendritic processes and cell body to cell size ratio. The novel analysis method provides a sensitive marker for microglial activation in the rat brain, which is quick and easy to perform and provides additional information about microglial morphology.