Police received a report from a man driving south along the expressway who saw an arm movement and a rock hurled near Flaxmill Rd bridge in Christie Downs just before 11.30pm.
The rock reportedly bounced on the road but missed the car.
No one was hurt.
Police deployed a helicopter to search the area but did not find anyone hiding in nearby bushes.
Officers monitored the expressway through the night.
INTERACTIVE MAP: Southern Expressway incidents
The latest incident marked the 11th rockthrowing event since January.
Rocks flung or dropped from footpaths and overpasses along the expressway had damaged vehicles and nearly caused injury to motorists.
Last month, Lauren Mangelsdorf and her two-year-old son Lachlan were driving near the Moore Rd overpass at Reynella when it was struck by a rock thrown from the side of the expressway.
Prime Minister Malcolm Turnbull on Sunday likened the dangerous act to starting a bushfire.