Members of the Transport Salaried Staffs’ Association (TSSA) union will walk out on Saturday and Sunday in a similar protest to the previous industrial action on December 30 and 31 last year.
Picket lines will be set up in Glasgow and Edinburgh, where striking workers will protest from 8am to 12pm on both days.
TSSA said publicly-owned ScotRail must resolve the dispute to avoid safety implications.
The union claimed the rail operator had offered no solution to ending the dispute despite discussions taking place, including an in-person talk with TSSA general secretary Maryam Eslamdoust.
Ms Eslamdoust, who will visit picket lines over the weekend, said: “Our members are vital to the safe running of Scotland’s railways day in, day out and deserve to be treated with respect by their employers over their legitimate grievances.
“Since our last walkout we have only grown in strength among these grades. We would prefer to have meaningful talks with ScotRail to move our dispute forward – however we go into these strikes united and determined.”
The dispute includes conductor team managers, driver team managers, on-train team managers and station team managers, operations development managers and senior operations development managers working at stations across Scotland.
ScotRail said it plans to run a full timetable over the affected dates.
As part of the dispute, TSSA members have withdrawn from ScotRail’s out-of-hours on-call period, which is generally between 11pm and 7am, during which it said ScotRail will have no first-line on-call coverage.
The dispute has been ongoing since December 2022 and is due to continue until early June this year.
On X, ScotRail said the strike action would have no impact on its services over the weekend, stating they would continue to run a “full timetable”.
A ScotRail spokesperson added: “This action will have no impact on services or customers.”
A Transport Scotrail spokesperson said: “While these negotiations are a matter for the employer Scotrail and the unions concerned, we have already been clear that we expect constructive talks to continue and a resolution to be reached as soon as possible.”
