Multiple factors can affect the synthesis of the prodrugs aminolaevulinic acid and its methyl ester to protoporphyrin. These may ultimately influence the efficacy of ALA-induced porphyrin as a photosensitiser for photodynamic therapy or fluorescence diagnosis. This study demonstrates the variation in total amount of porphyrin produced and cellular porphyrins synthesised in four different human cell lines after supplementation with these prodrugs. A non-invasive optical biopsy system was able to detect spectral changes associated with the more carboxylated porphyrins accumulating in oesophageal (OE19) and bladder (HT1197) carcinoma cells, and to a lesser extent neuroblastoma (SH-SY5Y) cells after a 24h incubation with the prodrugs. If the porphyrin profile changes during disease progression, or between normal and cancerous cells clinically, then the demonstrated non-invasive spectral analysis may be exploitable in distinguishing between normal, dysplastic and tumour tissue. Finally, the OE19 cell line was insensitive to photo-inactivation under the experimental conditions used, despite accumulating more porphyrin than the other cells lines.