Although aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) photodynamic therapy (PDT) is an effective FDA-approved therapy for actinic keratosis (AK), a substantial fraction of patients (up to 25%) do not respond to treatment. This study examined the feasibility of using pre-treatment measurements of PpIX concentration in AK lesions to predict response of ALA-PpIX PDT.A non-invasive fiber-optic fluorescence spectroscopy system was used to measure PpIX concentration in patients undergoing standard-of-care ALA-PDT for AK. All patients provided assessments of pain at the time of treatment (n = 70), and a subset reported pain and erythema 48–76 h after treatment (n = 13).PpIX concentration was significantly higher in lesions of patients reporting high levels of pain (VAS score ≥5) immediately after treatment vs. patients reporting pain scores below VAS = 5 (p < 0.022) (n = 70). However, pain was not an exclusive indicator of PpIX concentration as many patients with low PpIX concentration reported high pain. In a subpopulation of patients surveyed in the days after treatment (n = 13), PpIX concentration measured on the day of treatment was uncorrelated with pain-reported immediately after treatment (r = 0.17, p < 0.57), but positive correlations were found between PpIX concentration and patient-reported pain (r = 0.55, p < 0.051) and erythema (r = 0.58, p < 0.039) in the 48–72 h following treatment.These data suggest that in vivo optical measurements of PpIX concentration acquired before light delivery may be an objective predictor of response to ALA-PpIX PDT. Identification of non-responding patients on the day of treatment could facilitate the use of interventions that may improve outcomes.