Introduction: Neurosurgical 5-aminolevulenic acid (5-ALA) fluorescence-guided surgery (FGS) is evolving from highly subjective approach towards novel systems capable of quantifying the 5-ALA-induced protoporphyrin IX (PpIX) fluorescence. Optical tissue phantoms provide a cost-effective adjunct in obtaining data for the development of FGS systems. Material & Methods: Gelatin phantoms were prepared using variable concentration of PpIX and bovine blood in addition to changing pH level. Intralipid was used as the scatterer. A fluorescence spectrophotometer and novel aspirate tissue monitoring (ATM) device was used to measure the phantoms’ fluorescence emissions. Results: Increasing blood volume fraction (BVF) markedly reduced the PpIX fluorescence. Increments of 0.5% BVF reduced the fluorescence by 55–61% and 51–82% in milder and stronger concentrated phantoms, respectively. Increasing pH (6–8) augmented the fluorescence intensity in the ATM measurements by 50–130%. Conclusion: 5-ALA phantoms can streamline the development of new FGS systems and accelerate the transition from research to clinical practice.