The agreement will see the Potters and Orlando share best practice and business expertise, alongside promoting each other in their respective markets.
The two clubs, who have forged a close relationship in recent years, will liaise in terms of player recruitment and player development, as well as fan engagement and commercial aspects.
Interest in Premier League football in the US is at an all-time high thanks to coverage provided by NBC, who have rights to show all 380 top flight games per season live across its network and recently broadcast live from the Britannia Stadium for Stoke's game against Tottenham.
City Chief Executive Tony Scholes said: "It's an opportunity for us to share best practice with Orlando in a variety of areas, primarily in player recruitment, marketing and development, but also to give both clubs chance to grow in each other's markets.
"A large number of our fans already regard Orlando as their MLS side and I know that Stoke City are already followed by many Orlando fans.
"As an established Premier League club we are always looking at new ways to develop our profile overseas and our strategic partnership will help us to develop in the United States."
Orlando City Founder and President Phil Rawlins added: "It was very clear that we've always had a good relationship with Stoke City and it came down to us wanting to reignite that synergy and bring our brands closer together.
"This will be our only partnership in England, and we hope to ignite passions for both our clubs in each other's markets."