In this paper, we present an original method of dot pattern generation dedicated to large-size format light guide plate (LGP) design optimization, such as photo-bioreactors, the number of dots greatly exceeds the maximum allowable number of optical objects supported by most common ray-tracing software. In the proposed method, in order to simplify the computational problem, the original optical ... [Show full abstract] system is replaced by an equivalent one. Accordingly, an original dot pattern is splitted into multiple small sections, inside which the dot size variation is less than the ink dots printing typical resolution. Then, these sections are replaced by equivalent cells with continuous diffusing film. After that, we adjust the TIS (Total Integrated Scatter) two-dimensional distribution over the grid of equivalent cells, using an iterative optimization procedure. Finally, the obtained optimal TIS distribution is converted into the dot size distribution by applying an appropriate conversion rule. An original semi-empirical equation dedicated to rectangular large-size LGPs is proposed for the initial guess of TIS distribution. It allows significantly reduce the total time needed to dot pattern optimization.