The capabilities of two-dimensional separation using a high irradiance laser ionization orthogonal time-of-flight mass spectrometer (LI-O-TOFMS) were demonstrated in this paper. Ions were separated via their initial kinetic energy in one dimension and their mass-to-charge ratios in the other dimension. Investigation of the transient ion profiles after laser pulses revealed that the separation of analyte ions from multiply charged ions and gas species ions was achieved. Comparison of mass spectra in the normal accumulation mode and in the two-dimensional separation mode indicated that the relative sensitivity coefficients are stable and close to their true values in the two-dimensional separation mode, especially for trace elements that are prone to interference.