United will travel away to face the Northern Premier League Division One Midlands side, with a date and kick-off time for the fixture which will be played at the Sir Halley Stewart Field yet to be confirmed.
Scunthorpe progressed from the quarter-finals after beating Grantham Town 1-0 thanks to a late Mason O'Malley free-kick, while Spalding progressed after seeing off National League side Grimsby Town 2-0.
The competition has returned after not taking place last season due to the COVID-19 pandemic, with the Iron's last involvement prior to this season coming in 2019 when a youthful side was knocked out by Boston United in Round One.
The two sides last met in November 1980 to mark the opening of Spalding's new floodlights. The Iron won the game 6-0 on the night, with a brace from Dave Stewart and Steve Cammack, while Ian Botham and Stuart Pilling added the other two goals.
Scunthorpe are 20-time winners of the competition, but last picked up the accolade a decade ago in 2011.
The full draw for the semi-final is as follows:
Spalding United v Scunthorpe United
Gainsborough Trinity v Boston United