Police and protective service officers, who are known as PSOs and patrol the state's train system, working at the station in central Melbourne will strike for one hour at 11am on Friday.
They are pushing for a 24 per cent pay rise over four years and for an end to unpaid overtime.
The union says Friday's walkout is the biggest of 17 separate stoppages held in the past fortnight, with this round of industrial action marking the first time officers have gone on strike in more than two decades.
The force had more than 1000 vacancies and 700 workers were on long-term sick leave, Police Association secretary Wayne Gatt said.
"This is Victoria's largest police station," Mr Gatt said.
"It's the heartbeat of policing in Victoria and today it stops, because it, like all of our stations across the state, is at breaking point."
In May, an in-principle agreement was reached with officers for a nine-day fortnight and a 16 per cent pay rise over four years, but union members voted against the deal.
The force asked the Fair Work Commission to intervene, with a decision due by the end of 2024.
A Victoria Police spokesperson said the force respected the right of its members to take protected industrial action and it was waiting for the commission's ruling on how the dispute would be settled.
"Our police and PSOs do an amazing job each and every day and we agree they deserve to be well paid for the important work they do," the spokesperson said.
"Victoria Police remains committed to securing an outcome that recognises the challenges of policing and is fair for police, protective services officers and the Victorian community."
The action comes after NSW officers won a pay rise of at least 25 per cent in recent weeks, while Queensland Police offers a $20,000 relocation payments for recruits.
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