The pair were among a trio of players to miss from the spot in the European Championship final defeat to Italy last year, along with Jadon Sancho, who was not selected for the tournament in Qatar.
Rashford has been a fine performer for Southgate's side, scoring a trio of goals to sit joint-top of the scoring charts, while Saka netted a brace in the demolition of Iran in the opening group match.
Now into the knockout stage, with a round of 16 tie against Senegal on the horizon, the prospect of penalties continues to be a talking point – particularly regarding who could be called upon.
Asked if he had any reservations about either Rashford or Saka stepping up in a shootout, Southgate said: "None at all.
"Marcus' stats would be among the best in the world on penalty taking, and Bukayo is now taking them more regularly with his club. They're both in a good space with that."
In the World Cup in Russia, Southgate introduced players, including Rashford, off the bench in order to take penalties but was criticised for the same approach against Italy – with Rashford and Sancho both missing after their late introductions.
Southgate defended that tactic and hinted that similar moves could be made in the future, if required.
"In terms of the changes, Marcus came on as a sub in Moscow and took a penalty four or five minutes earlier than we put him on at Wembley," he added.
"I've seen lots of teams do that and scored, and it's the right thing. If you don't then it's wrong.
"We've got to stick with our processes, give ourselves the best percentages of winning, and we feel we are very well prepared.
"We've won two, we've lost the last one. We've refined some of those things we didn't think were quite right. We feel that the process is good."
Meanwhile, Senegal is hoping coach Aliou Cisse will clamber off his sick bed to be in the dugout for the knockout clash.
Cisse has been ill the last two days and was forced to miss a training session.
His assistant Regis Bogaert faced the press before the England meeting but remains confident Cisse will be on hand at Al Bayt Stadium.
"He's been sick for a couple of days now and he let us [the staff] take charge of training yesterday, with his instructions," he said.
"We are sure that at 10pm tomorrow, he will be there."
Senegal is African champion and edged out Ecuador to secure qualification from Group A and underline its growing reputation on the world stage, despite the absence of star striker Sadio Mane.
"We know Senegal can beat England. We need to focus on our African identity and be positive with the experiences we are having at the moment," Bogaert said.
"Since the last World Cup, we have matured. We can beat anybody and when we go on the pitch we need to be convinced of that."