Intending to improve the accuracy of determination of antibiotics, a two-dimensional diffusion method using large agar plate was introduced. Three antimicrobial agents, ampicillin, PC-904, and tobramycin, were used. Inhibition zones of B. subtilis on the agar plate were measured which were formed as a result of diffusion of these agents. The relationship between the concentration of antimicrobials and the size of inhibition zones was studied. Plotting the data-points on the graph, it was predicted that there might be a relationship of quadratic equation between the diameter of inhibition zone and the logarithm of concentration of the agents. On the other hand, mathematical considerations were taken to find out a physical principle or an equation which governs the diffusion of antibiotics in the agar. Assuming that antibiotics spreads in the agar after the principle of simple diffusion, an equation was lead which shows how the antibiotics distributes in the agar in relation to time. The equation was written as, (formula: see text) where, S is amount of antibiotics, D diffusion constant, r distance from the center of diffusion, t period during which the diffusion proceeds. As a result of the mathematical calculations mentioned, it was confirmed that the relation between the size of zones and the logarithm of concentration of antibiotics is described by a quadratic equation as predicted on the basis of experimental data.