Objective: In this work an irradiance and temperature controlled in-vitro system for conducting investigations in PDT and phototherapy is presented. Background data: The development of new light sources has caused a considerable increase in research and application of several photodynamic (PDT) therapeutic methods, as well as other light-based therapeutic techniques. However, further work is needed to fully understand and elucidate the mechanisms as well as to increase the effectiveness of PDT. Nowadays, there are no commercial systems to perform automated light exposure experiments with cultured cells. Also, there are very few reports of similar photoirradiation systems. Materials and methods: The system is composed of 24 independent light-emitting diodes that can be used to irradiate separate wells in a microwell plate. The system includes a module to measure changes in temperature within each irradiated well without contact. The light sources are placed on a plate that can easily be changed in order to irradiate at different wavelengths. The performance of the system is fully controlled with a computer, and all the experimental data are properly recorded. Results: The design, construction, operation, and a full characterization of the system are presented. Conclusions: A novel fully automated photoirradiation system has been developed. The system allows the design of the experiments in this area with precise dosimetry, temperature, and irradiation regime controls reducing manipulation of the samples and saving time.