The results of many investigations on low-level laser therapy are contradictory and this is due to the large number of illumination parameters as well as the inability to measure the possible effects after irradiation with the necessary objectivity and the fact that the light needs to pass thorough barriers (usually the plastic of the culture dish/plate and culture medium) to reach the cells. In this manner, the objective of this study was to determine the absorption coefficient, penetration depth and effective transmission in materials commonly used in cell cultures. Among the most commonly used wavelengths in low-level laser therapy, the lowest absorption coefficients were reached by DMEM and RPMI (α = 0.03 cm−1), from 633 to 690 nm, which reach an effective transmission of 93% of incident radiation and penetration depth of 33 cm. Among the solid materials in the same range of the electromagnetic spectrum, the lowest absorption coefficient was obtained for the polystyrene (Petri dish and well plate), with α = 1.31 cm−1, 78% of effective transmission and 0.76 cm of penetration depth. This article also presents a simple equation for estimating the amount of energy that will actually reach the sample.