!Beakman (Paul Zaloom)
* AnimeHair
* IAmNotSpock: Compare Beakman, the child-friendly scientist, to his actor, Paul Zaloom, the VERY adult political puppeteer.
* MadScientist
* StudentAndMasterTeam

!Lester the Rat (Mark Ritts)
* BecomingTheMask: In early episodes, he resented having to wear the rat costume. As {{Flanderization}} kicked in, he apparently began to actually identify himself as a man in a rat suit as if it were his species(as well as the fact that friends and family members inexplicably became people in rat suits as well).
* BigEater
* ClassicallyTrainedExtra: Started out this way when the show began.
-->'''Lester''': [[{{Hamlet}} "To be, or not to be?"]] ''That'' is the question, not "what is snot?"!
* DeadpanSnarker: He would be one if he wasn't constantly proven wrong.
* ExtremeOmnivore: Right along with BigEater.
* {{Flanderization}}: As the series progressed, the [[ClassicallyTrainedExtra disgruntled-actor-in-a-rat-suit]] bit was dropped in favor of Lester's more disgusting traits.
* GenreSavvy: Lester would occasionally succumb to the inevitable and inform Beakman that he was not going to pooh-pooh the latest Challenge, because he knew from experience Beakman was going to turn out to be right and make Lester look foolish in the process.
* TheLabRat: Subverted, despite being the guy in the lab in the rat suit.

!The Assistants
* AudienceSurrogate / TheWatson: Beakman's assistants help think through the answer, ask the obvious questions, and generally keep Beakman moving toward a response to the readers' questions.
* GenkiGirl: All three were highly energetic, particularly when rushing in from off-screen or during the intro to Beakmania.


!Josie (Alanna Ubach)
* AwesomeMomentOfCrowning: The last segment of Josie's last episode shows kids how they can do their own research (using the question "How do fireworks make those neat colors?" as an example). Beakman points her to resources, but Josie does most of the legwork and provides the final answer herself.
* PrecociousCrush: Josie is often awe-struck by Beakman's knowledge.

!Liza (Eliza Schneider)
* FingerlessGloves
* HospitalHottie / {{Meganekko}}: The "Dr. Liza" segments.
* ReplacementScrappy: Liza gets some hatred from fans for not being Josie, but she gets it fairly easy compared to Phoebe.

!Phoebe (Senta Moses)
* TheCastShowoff: Senta's martial arts training was put to good use in a segment about breaking boards.
* QuirkyCurls
* RapidFireInterrupting: At least a couple of times per episode, which only added to her "bratty know-it-all" status.
-->'''Beakman''' ''(after two of these interruptions back to back)'': Okay, Phoebe: Since you know ''every''thing...
* ReplacementScrappy: She's hated by fans even more so than Liza, due to the fact that she's not Josie ''or'' Liza.
* SchoolNurse: Nurse Phoebe. Most definitely the Western version.

!Don and Herb (voiced by Alan Barzman and Bert Berdis)
* BookEnds: Each episode begins and ends with a scene of them.
* EverythingsBetterWithPenguins
* GreekChorus
* ReactionShot: Often get one when a piece of information given in the show relates to them somehow.
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