Almost 3,000 people have signed a petition to protest Madonna's concert in the Czech capital.
Protest organizer Jiri Styler says local residents are afraid of traffic chaos and noise.
The outdoor venue is located on a field between a major highway and a large residential area. Intense security measures, including road closures and transport disruptions are in place before Thursday's show.
Local authorities said the show must be over by 10 p.m. and organizers will clean up the area, It is being used for a concert for the first time.
Styler said the protesters "are horrified" that more big concerts could follow in the area.
Some 40,000 are expected to attend the show, part of Madonna's "Sticky & Sweet" tour.