!Beakman (played by Paul Zaloom; voiced by Juan Alfonso Carralero [Mexican Spanish])
* AnimeHair
* MadScientist
* StudentAndMasterTeam

!Lester the Rat (played by Mark Ritts; voiced by Creator/SalvadorDelgado [Mexican Spanish])
* BecomingTheMask: In early episodes, he resented having to wear the rat costume as he was a professional actor whose agent booked him a crummy role on a kids' edutainment show. 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.
* 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 (played by Alanna Ubach; voiced by Creator/LauraTorres [Mexican Spanish])
* 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 (played by Eliza Schneider; voiced by [[Creator/RocioPrado Rocío Prado]] [Mexican Spanish])
* FingerlessGloves
* HospitalHottie / {{Meganekko}}: The "Dr. Liza" segments.

!Phoebe (played by Senta Moses; voiced by [[Creator/RocioPrado Rocío Prado]] [Mexican Spanish])
* 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...
* SchoolNurse: Nurse Phoebe. Most definitely the Western version.

!Don and Herb (voiced by Alan Barzman and Bert Berdis [original], Alejandro Villeli and Octavio Rojas [Mexican Spanish])
* 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.