Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is a novel treatment method with much potential benefit for cancer detection and eradication. Formulation into drug delivery systems, such as aqueous solutions and emulsion based creams is complicated by its rapid dimerisation to pyrazine 2,5-dipropionic acid (PY); a compound with scant documentation in terms of toxicity and effect during PDT. This degradation is especially noticeable, where pH is adjusted upwards to avoid local irritation. A good case in point is bladder instillation of ALA for treatment and diagnosis of urothelial neoplasia. This work describes a rapid and validated HPLC method designed to assess the formation of PY in ALA-loaded vehicles. PY eluted as a single peak (Rt=5.0 min) with good intra- and inter-day reproducibility and limits of detection and quantification found to be 0.01 and 0.04 microg ml(-1), respectively. Sample stability for upto 16 h was demonstrated, allowing autoinjection cycles to be performed. PY formation was detected in typical buffers used for bladder instillation after 6 h of storage, emphasising the need to use these preparations immediately upon manufacture if intended for photodynamic purposes. Moreover, upto 2.35% (w/w) PY was detected in artificial urine after 6 h storage at ambient temperature indicating that formation in vivo is likely to occur once bladder instillations are in situ and exposed to endogenous urine. As a result, ALA instillation times should be kept to the minimum needed for safe and successful treatment or diagnosis. PY extraction from semi-solid devices approached 100% efficiency demonstrating that the reported assay is suitable for evaluating stability of novel dosage forms intended for ALA delivery.