And the former Wales captain is aware they need to deliver when the host the Dragons in a Welsh derby fixture on Saturday.
"The Glasgow match was a hugely disappointing defeat and display for us," said Humphreys.
"We should not take anything away from Glasgow because they were outstanding in the second-half and probably played the best they have all year.
"But the difference in the level of the performance to a week before is not good enough for the ambitions that we have here.
"Mentally we weren't where we should have been and it is something we have to confront.
"We won't hide from that and we expect more from the players because we had a good enough side to go there and win.
"If our ambitions are to win this league and defend it like the champions that we are then can't have the big drop-off in performance.
"We build ourselves up to huge games but the next weekend we can't afford not to deliver."
With the autumn international window just around the corner, the Ospreys have lost 17 players to respective national sides and Humphreys knows his squad is going to be fully tested over the next few weeks.
"There is going to be a huge emphasis on the group of players that are left here who don't go away with Wales," he added.
"People ask why we have a big squad but we are losing 17 players to international rugby.
"This is the opportunity for the rest of the squad to show they should be challenging for the first team. They need to stand up and be counted. .
"We are delighted we have so many boys in the international scene and it makes us coaches having to earn our money during this period in time, but it is a challenge."
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