Abstract δ-Aminolevulinic acid–photodynamic therapy (ALA-PDT) has emerged as a useful technique in the treatment of superficial basal cell carcinoma, actinic keratosis, squamous cell carcinoma and tumors of other organs. Earlier reports mention that there is reappearance of protoporphyrin IX (PpIX) after photoirradiation of tumors. This property of reappearance of PpIX is being utilized to treat nodular tumors by fractionated light dose delivery. However, there is still no unanimously accepted reason for this reappearance phenomenon and the rate of resynthesis after PDT. On account of this, studies are carried out on the estimation of the pharmacokinetics of the ALA-induced PpIX in mice tumor models and the surrounding normal tissues before and after PDT. Further, a mathematical model based on a multiple compartment system is proposed to estimate the rate parameter for the diffusion of PpIX from the surrounding normal tissues into the tumor tissue (km) caused by photobleaching during PDT with irradiating fluences of 36.0 and 57.6 J/cm2. The km value at two different fluences, 36.0 and 57.6 J/cm2, are estimated as 3.0636 ± 0.7083 h−1 and 6.9231 ± 2.17651 h−1, respectively. Further, the rate parameter for the cleavage and efflux of ALA (k1) and the rate parameter for the evasion of PpIX from the tumor tissues after PDT (kt) were also estimated by fitting the experimental data to the developed mathematical model. The statistical significance of the estimated parameters was determined using Student's t-test. The experimental results and the rate parameters obtained using the proposed compartment model suggest that in addition to the earlier reported reasons, the invasion or diffusion of PpIX from the surrounding tissues to the tumor tissues after photoirradiation might also contribute to the reappearance of PpIX after PDT.