Nanocrystalline cellulose (CNC) consists of cellulose polymers arranged in a highly ordered and uniform structure, resulting in coherent interactions with electromagnetic waves, offering significant potential in advanced optical applications due to its intrinsic anisotropy and unique chiral properties. Sulfated CNC (CNC-SO3H) stands out for its surface charge, colloidal stability, and ability to self-assemble into films with optical activity. To optimize CNC films for applications in optical devices and sensors, it is essential to understand and develop a scientific understandning on controlling the factors governing crystallite alignment and the influence of surface modifications.This work presents the alignment of CNC crystallites by employing linear dichroism (LD) and circular dichroism (CD) spectroscopy, with additional characterization using Mueller matrix polarimetry (MMP) at Beamline 23 of Diamond Light Source. CNC-SO3H films were prepared with two amines, 3,3′-iminodipropanenitrile, and diethanolamine, which coordinate with sulfate groups as ion pairs, and by azetidinium conjugation. The films, derived from 1 wt% CNC-SO3H suspensions, were analyzed to assess the impact of these additives on crystallite orientation.LD and CD measurements revealed distinct differences in alignment between pristine and modified CNC films. CNC-SO3H exhibited a predominantly positive CD signal, while amine-modified systems displayed varied CD patterns. Diethanolamine resulted in a uniform CD distribution, whereas dinitrile-modified CNC exhibited a mix of positive and negative signals. Upon azetidinium conjugation, dinitrile-modified films showed enhanced uniformity and higher CD signals compared to diethanolamine.LD analysis further confirmed differences in alignment, with pristine CNC showing a homogeneous pattern, while amine-modified samples exhibited reduced alignment. Diethanolamine-modified films maintained higher alignment compared to dinitrile-modified ones. These findings demonstrate the effectiveness of LD and CD in characterizing CNC crystallite orientation and highlight the influence of surface modifications on optical properties.