Quantification of 5-aminolevulinic acid (ALA) in solution, and methods used to achieve this, have been extensively reported in the literature. However, validated methods have only rarely been presented and never have methods been compared. Due to a necessity in drug delivery research for optimised and validated methods for determination of ALA in solution, this paper compares, for the first time, two such methods validated to International Conference on Harmonisation (ICH) standards. Of major importance, derivatisation of ALA with acetyl acetone and formaldehyde was found to be more suitable for routine fluorimetric HPLC analysis of ALA than derivatisation with o-phthaldialdehyde and 2-mercaptoethanol. This former method was successfully utilised in the comparison of in vitro drug release from a proprietary ALA cream and a novel bioadhesive patch-based system. In addition, determination of ALA in tissue is necessary to compare different topical formulations, in terms of their ability to deliver the drug successfully, and different tissue types, to assess their barrier properties to penetration of the drug. Consequently, this paper also describes the use of liquid scintillation spectroscopy as an analytical tool for rapid, convenient and routine quantification of ALA in tissue and determination of penetration depth following topical application of creams and patches.