Background 5-Aminolevulinic acid (5-ALA) has become an important assistant in glioblastoma (GB) surgery. Unfortunately, its price affects its widespread use. Objective The aim of this study was to compare commercial 5-ALA with the pharmacy-compounded solution. Methods Using first an in vitro experimental approach, different concentrations of the pharmacy-compounded solution and commercial 5-ALA were tested in U87MG, LN229, U373, and T98G commercial glioblastoma cell lines. Fluorescence intensity was compared for each concentration by flow cytometry. Mean fluorescence of culture supernatant and lysate samples were analyzed. In a second phase, both preparations were used for surgical glioblastoma resection and tumor samples were analyzed by confocal microscopy. Mean fluorescence intensity was analyzed for each preparation and compared. Results There was a high variability of fluorescence intensity between cell lines, but each cell line showed similar fluorescence for both preparations (compounded preparation and commercial 5-ALA). In the same way, both preparations had similar fluorescence intensity in glioblastoma samples. Conclusion Both, compounded and commercial 5-ALA preparations produce equivalent fluorescent responses in human glioblastoma cells. Fluorescence intensity is cell line specific, but fluorescent properties of both preparations are undistinguishable.