Protonation and solvation of functionalized heterocyclic aromatic molecules, which often occur as biomolecular building blocks, are important processes in (bio-) organic biochemistry. Herein, we study the protonation and microsolvation mechanisms of 5-hydroxyindole (5HI, 1H-indol-5-ol, C8H7NO), the chromophore of serotonin, produced by electron and and and chemical ionization using infrared photo dissociation (IRPD) spectroscopy of mass-selected cold 5HIH(+)-L-n clusters (L = Ar/N-2, n <= 3) and dispersion-corrected density functional theory calculations (B3LYP-D3/aug-cc-pVTZ). Isomer-selective OH and NH stretch frequencies in the spectral range 3000-3800 cm(-1) reveal the coexistence of at least four protonated species: the most stable syn (cis) isomer protonated at the C3 position of indole, both syn- and anti-rotamers protonated at C4 of the phenol ring, and the drastically less stable O-protonated isomer (Delta E-0 = 117.1 kJ/mol) stabilized by kinetic trapping. Manipulation of the IRPD conditions (fragmentation channels) facilitates the spectroscopic isolation of O-protonated species. Upon protonation, the acidity of the OH group increases in the order 5HIH(+)(C3), 5HIH(+)(C4), and 5HIH(+)(O), while the acidity of the NH group decreases along this series, strongly affecting the microsolvation motifs of the individual isomers. Comparison of 5HIH(+)-L to the corresponding neutral and radical cation clusters reveals the impact of both protonation and ionization on the interaction with nonpolar ligands. Furthermore, our results are compared to protonated phenol, for which similar gas-phase protonation mechanisms have been found. Comparison of 5HIH(+)-L with the corresponding clusters of protonated phenol illustrates the effects of functional substitution and addition of aromatic rings on intermolecular potential.