Sheffield-born centre-back Maguire joined the Three Lions squad this summer having not played since May 9 because of ankle ligament damage.
Southgate said before the game the former Hull City and Sheffield United player would be “involved” against Scotland on Friday, but that involvement extended no further than being an unused substitute.
Midfielder Henderson’s only football since February was 45 minutes in a pre-tournament warm-up friendly at Middlesbrough.
But the pair captain England’s most high-profile clubs, Manchester United and Liverpool, and have contributed off the field if not on it, says Southgate.
“Both are training very well and both will be involved,” he said. “We’re happy with their progress.
“They’ve already had a massive impact around the training sessions and around the squad. I couldn’t be happier with the influence and impact our senior players have had on the group.
“Of course they want to get on the pitch and we’re hoping they can play a big part in this tournament for us.”
Less experienced players have stepped up in Maguire and Henderson’s place, making it harder for them to get gametime to be sharp for the knockout stages.
Leeds United’s Kalvin Phillips was outstanding in midfield against Croatia in the opening game, and it was no surprise he retained his place for the 0-0 draw against Scotland.
Left-sided centre-back Tyrone Mings was unconvincing in the Middlesbrough friendlies before the tournament, but very assured in both group matches.
Captain Harry Kane is under pressure after starting the competitions with two performance well below his usual high standards both in a footballing and physical sense, but Southgate has made it clear he will start against the Czech Republic tonight.
“I think it’s the world of a big player,” he reasoned. “Throughout the time I’ve been England manager I must have answered these questions about Harry on three or four periods of time and inevitably he’s come through those periods, scored goals for us and played incredibly well.”
Last night’s results confirmed England’s place in the knockout stages, giving Southgate more scope to give Maguire and Henderson playing time if he wishes.