Sevilla's 2-1 first-leg victory at the Nou Camp last week was enough to put the holders out of the competition despite Barca winning 1-0 at the Sanchez Pizjuan on Wednesday.
It is the first time Guardiola has experienced elimination from a competition since taking the helm at Barca last season and leading them to an unprecedented haul of six titles.
"We'll have to see how we react," Guardiola said after Wednesday's game.
"I feel I've let my players down. I couldn't have asked for any more from them. If we go out at the last 16 stage it is because something wasn't right and I haven't seen it." Barca continue to set the pace at the top of La Liga with 43 points from 17 matches, two points ahead of second-placed Real Madrid.
Waiting for any slip-ups are Barca's arch-rivals Real, who have a tough trip to the traditionally unwelcoming San Mames to face Athletic Bilbao on Saturday.
Coach Manuel Pellegrini will have to decide whether to play Raul or Karim Benzema up front after in-form Argentine striker Gonzalo Higuain tore a thigh muscle in last weekend's 2-0 victory over Real Mallorca.
"It's always a tough stadium to visit ... The fans support their team and roar like lions, but ... there are only 22 players on the pitch," goalkeeper Iker Casillas told reporters.
"There is nothing positive to take from the game," Valencia coach Unai Emery told reporters.
"Everyone's concentration needs to be at the maximum all the time to make sure what happened in the second half does not happen again."
