Magnetophoretic isolation of biochemical and organic entities in a microfluidic environment is a popular tool for a wide range of bioMEMS applications, including biosensors. An experimental and numerical analysis of magnetophoretic capture of magnetic microspheres in a microfluidic channel under the influence of an external field is investigated. For a given microfluidic geometry, the operating conditions for marginal capture is found to be interrelated in such a manner that a unique critical capture parameter [Pi(crit) = ((Iota(crit)a))(2)/Q(eta)], that is proportional to the ratio of the magnetic force to viscous force, can be identified. Influences of the flow rate, magnetic field and other parameters on the particle trajectories in the microfluidic channel are investigated both numerically and through bright-field imaging under a microscope. Like the event of critical capture, particle trajectories are also found to be guided by a similar parameter, pi. Magnetophoretic capture efficiency of the device is also evaluated as a function of a nondimensional number [Pi(*) = chiP(2)a(2) / (U(null)etah(5)], when both numerical and experimental results are found to agree reasonably well. Results of this investigation can be applied for the selection of the operating parameters and for prediction of device performance of practical microfluidic separators.