Hypothesis: Rodlike cellulose nanocrystals (CNCs) exhibit significant potential as building blocks for creating uniform, sustainable materials. However, a critical hurdle lies in the need to enhance existing or devise novel processing that provides improved control over the alignment and arrangement of CNCs across a wide spatial range. Specifically, the challenge is to achieve orthotropic organization in a single-step processing, which entails creating non-uniform CNC orientations to generate spatial variations in anisotropy. Experiment: A novel processing method combining frontal ultrafiltration (FU) and ultrasound (US) has been developed. A dedicated channel-cell was designed to simultaneously generate (1) a vertical acoustic force thanks to a vibrating blade at the top and (2) a transmembrane pressure force at the bottom. Time-resolved in situ small-angle X-ray scattering permitted to probe the dynamical structural organization/orientation of CNCs during the processing. Findings: For the first time, a typical three-layer orthotropic structure that resembles the articular cartilage organization was achieved in one step during the FU/US process: a first layer composed of CNCs having their director aligned parallel to the horizontal membrane surface, a second intermediate isotropic layer, and a third layer of CNCs with their director vertically oriented along the direction of US wave propagation direction.