An approach which could accurately decompose deflection of prismatic beams into flexural and shear deflections was proposed. Equation for estimation of decomposed flexural deflection was derived, and changes of diagonals in length caused by those experimentally measured longitudinal deformations were developed based on small deformation theory and under the assumption that flexural deflection can be represented by longitudinal deformation of upper fiber and lower fiber along prescribed beam segment. Equation for estimation of decomposed shear deflection was derived in terms of experimentally measured diagonal deformations under the assumption that shear deformation can only change length of diagonals of prescribed beam segment. Formula for estimation of deflection caused by rotation of target section was derived in terms of experimentally measured target section rotation for asymmetrical load condition. The fore mentioned decomposed flexural deflection, decomposed shear deflection and deflection caused by measured target section rotation add up to estimated deflection of target section, which is completely independent of directly measured deflection at target section. Loading tests on four prismatic rectangular reinforced concrete beams were conducted. Longitudinal deformation of upper fiber and lower fiber along shear span, diagonal deformations and rotation of load point section were measured. Estimated deflection of load point section calculated by using those measured data accorded with directly measured deflection at each load level, which verified the reliability of the proposed deformation decomposition approach.