The Masters and US Open champion had toppled McIlroy from the number one ranking two weeks ago with a runner-up finish to Jason Day at the PGA Championship.
But in his first tournament since then, he endured two difficult days at Plainfield Country Club, where he followed up a first-round 74 with a 73 on Friday to miss the cut by five strokes with a seven-over total of 147.
Two-time Masters champion Bubba Watson was the halfway leader of the $8.25 million tournament, the first leg of the US PGA Tour playoffs.
Watson rolled in a 12-foot birdie putt at the 18th to cap a two-under 68 for a seven-under total of 133.
He separated himself from a quartet of players on 134 that included British Open champion Zach Johnson, who had five birdies without a bogey in a 65.
Johnson was joined at six-under by Sweden's Henrik Stenson (66) and Americans Tony Finau (69) and Jason Dufner (68).
But it was Spieth in the spotlight as the 22-year-old Texan tried to rebound from a first round that marked his worst score in relation to par all season.
He had confidently vowed to bounce back, but couldn't gain any traction as he followed a first-hole birdie with back-to-back bogeys at the fourth and fifth. Another birdie at the sixth was promptly followed by two more bogeys at the seventh and eighth.
Spieth rallied briefly with birdies at 10 and 11, but ended up with a double-bogey seven at the par-five 12th where he incurred a penalty for stepping on his ball while he was looking for it in a grassy hazard.