The initial fixation of cementless tibial trays after total knee arthroplasty is crucial to bony ingrowth onto the porous surface of the implants, as micromotion magnitudes exceeding 150 μm may inhibit bone formations and limit fixation. Experimental measurement of the interface micromotions is still very challenging. Thus, previous studies investigated micromotions at the bone-tray interface via finite element methods, but few performed direct validation via in vitro cadaveric testing under physiological loading conditions. Additionally, previous models were validated by solely considering relative displacements of the marker couples placed around the tray-bone interface. In this paper, we present an experimental-computational validation framework for investigating micromotions at the tray-bone interface under physiological conditions. Three cadaveric specimens were implanted with cementless rotating-platform implants and tested under gait, deep knee bending, and stair descent loads. Corresponding subject-specific finite element models were developed and used to predict the marker (tray-bone) relative displacements and tibial surface displacements. Experimental measurements were used to validate model estimations. Subsequent sensitivity analyses were performed on implantation and friction parameters to represent model uncertainties.The models appropriately differentiated between locations, activities, and specimens. The average root-mean-square (RMS) differences and correlations between measured marker relative displacements and predictions from the ‘best-matching’ models were 13.1 μm and 0.86. RMS differences and correlations between measured surface displacements and predictions were 78.9 μm and 0.84. Full-field interface micromotions were investigated and compared with predicted marker relative displacements. The marker relative displacements underestimated the actual interface micromotions. Initial tray-bone alignment in anterior-posterior, flexion-extension, and varus-valgus degrees of freedom have a considerable impact on the interface micromotions. The validated cadaveric models can be further used for pre-clinical assessments of new TKR tray design. The outcomes of the sensitivity analyses provide further insights into reducing interface micromotions via clinical techniques.