The League One side had won their previous five games at Saltergate but Orient had not lost on their travels and first half goals settled the replay in their favour.
John Mackie and Shane Tudor scored either side of a Kevan Hurst strike and although they had to defend in the second half, the O's deserved to go through.
The League Two leaders were the sharper side from the start and played some composed football before they went in front in the 18th minute.
Barry Roche only succeeded in punching a free-kick out to Mackie who placed a side-foot shot into the unguarded net from 20 yards.
Chesterfield had not figured as an attacking force but in the 37th minute a good cross from Colin Larkin was crashed into the roof of the net with the help of a deflection by Hurst.
Chesterfield have made a habit of coming from behind to win this season but it was Orient who struck the next blow when Tudor restored their lead two minutes before the interval.
A superb cross-field pass from Matthew Lockwood found Tudor on the right and he went inside Shane Nicholson before crashing a shot past Roche and into the net via the post.
It was a superb goal but Orient had to show quality in and around their own penalty area in the second half.
Glyn Garner saved from Larkin with his legs and Wayne Allison just failed to connect with a low cross from Alex Bailey in the 63rd minute.
But Chesterfield's best opening fell to Derek Niven who shot high over the bar after Hurst had another fierce effort beaten out.
Although the home side kept pressing forward they did not create another clear chance, although Tudor made a superb tackle on substitute Adam Smith near the end.
This was yet another FA Cup disappointment for Chesterfield who have now failed to win a replay since 1980, but Orient must now fancy their chances of making the third round.