5-Aminolaevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy (PDT) as precursors of the photosensitizer, protoporphyrin IX (PpIX) in dermatology and urology. However, ALA–PDT is limited by the low bioavailability of ALA due to the fact that ALA is poorly absorbed by cells by virtue of its zwitterionic nature at physiological pH. In order to improve the therapeutical effect and induce higher levels of PpIX, a series of ALA prodrugs were synthesized by the conjugation ALA to 3-hydroxypyridin-4-ones (HPO) iron chelator using an amino acid linkage via amide bonds. Pharmacokinetic studies indicated that one ALA–HPO conjugate significantly enhanced PpIX production in a range of tumor cell lines over above that caused by ALA alone or the co-administration of ALA and CP94 (1,2-diethyl-3 hydroxypyridin-4-one). The intracellular porphyrin fluorescence levels showed good correlation with cellular photo-toxicity following light exposure, suggesting the potential application of the ALA–HPO conjugates in photodynamic therapy.