Acyclovir (ACV) is an antiviral drug used for the treatment of herpes simplex virus infection (HSV) and varicella zoster virus infection (VZV). One of the problems in acyclovir formulation is low solubility in water. The objective of this study is to find out the influence of milling time toward inclusion complex’s character that being formed between ACV and β-cyclodextrin (β-CD). The milling process used ball mill with variation of time in 1 hour, 3 hours, 6 hours. Preparation of inclusion complex used kneading method in 1:1 molar ratio. The characters of ACV- β-CD inclusion complex observed by using X-ray Powder Diffraction (XRD), Differential Thermal Analysis (DTA), Fourier Transform Infra-Red (FTIR), Scanning Electron Microscopy (SEM) and dissolution profile. In the dissolution profile observed a decreasing of dissolution rate on milled ACV inclusion complex compared with ACV inclusion complex without milling. Milling process create aggregation which is decreases the dissolution rate of inclusion complex.