A method of controlling fluid flow within a microfluidic circuit (10) using external valves and pumps connected to the circuit (10) is disclosed. The external valves and pumps, which are not a part of the microfluidic substrate (11), control fluid pumping pressure and the displacement of air (17) out of the fluid circuit (10) as fluid enters into the circuit (10). If a valve is closed, air (17) cannot be displaced out of circuit, which creates a pneumatic barrier (18) that prevents fluid from advancing within the circuit (under normal operating pressures).