Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway, from the substrate aminolevulinic acid (ALA), and can be used for tumor detection, monitoring or photodynamic therapy. PpIX production varies considerably between tumor cell types, and determining the cell types and methods to optimize production is a central issue in properly utilizing this drug. A panel of eight cancer cell types was examined for PpIX production capacity, including breast, prostate, and brain cancer tumors, and the production varied up to 10-fold among cell types. A positive correlation was seen between mitochondrial content and naturally occurring PpIX prior to ALA administration, but mitochondrial content did not correlate to the yield of PpIX resulting from the addition of ALA. Interestingly, total cell size was positively correlated to the yield of PpIX from ALA administration. Addition of an iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed, thereby further increasing the yield of PpIX. Those cell types that had the lowest ALA to PpIX production without L1 showed the largest percentage increase in production with L1. The study indicates that use of L1 in tumors with a lower innate production of PpIX with ALA alone may be the most productive approach to this combined delivery.