5-aminolevulinc acid (5-ALA)-based photodynamic therapy (PDT) and photodiagnosis (PD) present many advantages over treatments with conventional photosensitizers (PS). It offers great tumor specificity, reduced photosensitivity reactions caused by PS accumulation in non-targeted tissues and also inherent PS metabolism into endogenous non-fluorescent heme. However, chemical instability, low bioavailability and poor pharmacokinetic profile limit systemic efficacy of 5-ALA. Here, we present a comprehensive in vitro evaluation of novel phosphatase-sensitive prodrugs of 5-ALA. These prodrugs are designed to be activated by ubiquitously expressed phosphatases with much improved chemical stability and reduced acute toxicity profile. PpIX kinetic measurements and flow cytometry show accumulation of PpIX upon incubation with phosphatase-sensitive prodrugs in PC3 human prostate cell cancer, MCF7 breast adenocarcinoma, U87MG glioblastoma, T24 bladder cancer and A549 lung carcinoma cells. They revealed a different fluorescence kinetics and dose-response curves for the different types of 5-ALA prodrugs. These experiments have allowed us to identify the most promising cancer cell types for phospho- 5-ALA prodrugs. Confocal fluorescence microscopy provided further evidence of fluorescent protoporphyrin IX accumulation and sub-cellular localisation. These findings, together with the low toxicity profile of phosphatase-sensitive prodrugs of 5-ALA and good response to PDT provide solid basis for future translational development in PC3, MCF7 and U87MG cancer types.