Identification of operating deformation processes and assessment of the resulting strain partitioning are critical concerns for mechanical properties prediction and microstructure optimization in complex alloys such as α/β titanium alloys. Lattice rotation relative to the initial orientation was presently used as a marker of slip activity. A Ti-6Al-4V specimen with a bi-modal microstructure was tested in tension in a scanning electron microscope. Crystallographic orientations were characterized in situ using electron back-scattered diffraction (EBSD). A successful prediction of activated slip systems was achieved using the rotation axis associated with plastic activity. The combination of this procedure and slip traces analysis offers an insight into the determination of both slip plane and slip direction of active slip systems. Based on classical crystal plasticity formulations, the magnitude of the rotation relative to the initial orientation was interpreted in terms of plastic shear magnitude. A quantitative assessment of plastic strain at the microstructure scale was then carried out using lattice rotation data. This approach enabled to discuss strain partitioning in Ti-6Al-4V considering the influence of microstructural features and active slip modes.