The Reveal that Trixie is the teacher at the "hospital school" Twilight has to attend and the ongoing frustration that leads to. Especially as she turns out to be an awesome teacher everyone, especially Pinkie, enjoys, but Twilight.
Twilight: Does she do magic tricks during every lesson.
Silver Glow's introduction and the reveal that she thinks this is a spa are also amusing.
Twilight, Applejack, and Pinkie Pie's first breakfast together after Twilight wakes up in the Asylum, is a surprisingly light hearted-mostly-and funny scene, compared to the rest of the story.
As Rainbow Dash is about to call quits on Twilight's plan, Fluttershy finally speaks, if only for a moment, to stop her in chapter twenty seven. Twilight is left so speechless, she can't bring herself to say another word to either of them, before they leave.
One-Scene Wonder: Silver Glow, despite only appearing twice so far, her introduction was very memorable.
Pretty much every backstory of any character is this.
Twilight being told that Spike was just a figment of her imagination in "The Asylum Verse". It definitely doesn't help that her friends are excited at her revelation, that her trusted assistant/adopted little brother wasn't real. Not that they mean to upset her.
The reveal that Sweetie Belle was lobotomized and if Twilight's treatment fails, she will likely suffer the same fate.
The Woobie: Pretty much everyone in Broad Hoof Psychiatric Hospital counts, considering the type of place it is.