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Vertical deflection of a frame beam is an important indicator in the limit-state analysis of frame structures, particularly for steel–concrete composite beams, which are usually designed with large spans and heavy loads. In this study, the equivalent flexural stiffness of composite frame beams is analysed to evaluate their vertical deflection. A theoretical beam model with a spring constraint boundary and varied stiffness segments is established to consider the influence of both the rotation restraint stiffness at the beam ends and the cracked section in the negative moment region, such that the inelastic bending deformation of the composite beams can be elaborately described. By an extensive parametric analysis, a fitting formula for evaluating the equivalent flexural stiffness of the composite beams, including the effects of the rotational constraint and the concrete cracking, is obtained. The validity of the proposed formula is demonstrated by comparing its calculation accuracy with those of existing design formulas for analysing the equivalent flexural stiffness of the composite beam members. Moreover, its utility is further verified by conducting non-linear finite element simulations of structural systems to examine the serviceability limit state and the entire process evolution of beam deflections under vertical loading. Finally, to facilitate the practical application of the proposed formula in engineering design, a simplified method to calculate the deflection of composite beams, which utilises the internal force distribution of elastic analysis, is presented based on the concept of equivalent flexural stiffness.