Lower league sides Ayr United, Dundee and Falkirk will undergo Covid-19 tests before their Scottish League Cup ties against Premiership teams.
Ayr face Livingston, Dundee meet Hibs and Falkirk take on Rangers his month.
However, teams not facing Premiership opposition will not be required to test while top-flight clubs will continue their regular testing for meeting their league rivals in the last 16.
This extends the SPFL’s testing regime from the first-round group stage.
There are three all-Premiership ties and two all-Championship matches in round two.
As with the first round, the SPFL say any team “unable or unwilling” to field a team will forfeit the match on the basis of a 3-0 defeat and if both teams in a tie do not fulfil the fixture, lots will be drawn to decide who progresses to the quarter-finals.
SPFL chief executive Neil Doncaster commented: “Given the fact that the fixture calendar this season is the tightest in living memory, we have had to take unprecedented steps to ensure that the Betfred Cup matches have definitive results based on the original fixture dates.
“There will undoubtedly continue to be challenges as we progress through the season, but everyone in the game is determined to play their part in ensuring that the best possible football, in the safest possible conditions, is delivered across all divisions and in every competition.”
Last 16 ties
Saturday 28 November (15:00 unless stated)
Alloa v Hearts
Arbroath v Dunfermline
Hibernian v Dundee
Livingston v Ayr United
Motherwell v St Johnstone
St Mirren v Aberdeen (17:15)
Sunday 29 November
Celtic v Ross County (14:00)
Falkirk v Rangers (17:00)