Portsmouth will compete in the third tier of English football for the first time in 30 years after their relegation from the Championship was confirmed.
Pompey striker Luke Varney slotted in Marko Futacs's pass to cancel out Jake Buxton's first-half opener for Derby.
But Steve Davies converted a winning penalty four minutes later to deny Michael Appleton's side a small consolation after a torrid season.
Bristol City's home win against Barnsley sealed Portsmouth's fate.
It has been a terrible season for the South Coast club, who were docked 10 points in February after going into administration.
The club still owes £58m and Trevor Birch, of administrators PKF, said earlier this month liquidation was still a real possibility because he has received no firm offers to buy the club.
Despite the uncertain future, Appleton and his players had hoped to finish their home campaign on a high, but they fell behind when an unmarked Buxton headed in Jamie Ward's right-wing cross from close range.
With news of Bristol City's progress at Ashton Gate filtering through, the home fans tried to rally their side and Scott Allan fizzed a shot against the crossbar.
Portsmouth finally got their reward with 16 minutes to play through Varney, but their joy was short-lived when Ward was brought down by Jason Pearce in the box.
Despite Davies' spot-kick striking keeper Jamie Ashdown's legs, it still found its way in as Derby held on to win their final away fixture of a league campaign for the first time in 10 years.
But the overwhelming emotion at Fratton Park was one of dejection as Portsmouth contemplated life in League One next season, less than four years after winning the FA Cup at Wembley.