Deep inelastic scattering experiments at SLAC in the 1970's confirmed that quarks exist and that they have fractional charges. See this from which the following quote is taken:
These properties were so odd that for a number of years it was not
clear whether quarks actually existed or were simply a useful
mathematical fiction. For example, quarks must have charges of + 2/3e
or - 1/3e, which should be very easy to spot in certain kinds of
detectors; but intensive searches, both in cosmic rays and using
particle accelerators, have never revealed any convincing evidence for
fractional charge of this kind. By the mid-1970s, however, 10 years
after quarks were first proposed, scientists had compiled a mass of
evidence that showed that quarks do exist but are locked within the
individual hadrons in such a way that they can never escape as single
entities.
This evidence resulted from experiments in which beams of electrons,
muons, or neutrinos were fired at the protons and neutrons in such
target materials as hydrogen (protons only), deuterium, carbon, and
aluminum. The incident particles used were all leptons, particles that
do not feel the strong binding force and that were known, even then,
to be much smaller than the nuclei they were probing. The scattering
of the beam particles caused by interactions within the target clearly
demonstrated that protons and neutrons are complex structures that
contain structureless, pointlike objects, which were named partons
because they are parts of the larger particles. The experiments also
showed that the partons can indeed have fractional charges of + 2/3e
or - 1/3e and thus confirmed one of the more surprising predictions of
the quark model.
While it is true that we cannot seperate a single quark from a proton due to the color confinement property of the strong color force, it turns out that another property of the strong color force, asymptotic freedom, allows very high energy deep inelastic scattering to probe the properties of "free" quarks. These deep inelastic scattering experiments of electrons on protons established that there really were point like constituents of protons that had fractional electric charge and thus validated the quark model of hadrons.
