Lyotropic chromonic liquid crystals have great potential in both biosensing and optical devices due to their biocompatible nature and strong optical characteristics. These applications, however, demand a homogeneous and stable alignment on anisotropic surfaces, a challenge that, so far, has not been solved adequately. In this work, it is shown how to drastically increase the quality of in-plane alignment and stability of these liquid crystals on conventional rubbed polyimide substrates by the addition of a small amount of a nonionic surfactant. Samples with surfactant show excellent alignment that is stable for months, while control samples without surfactant show much poorer alignment that further deteriorates in days. Also, well-aligned dry films of chromonics can be prepared following this approach. It is demonstrated how to obtain high-quality alignment by controlling the concentration and the nature of the surfactant, in particular its molecular structure and hydrophilic/ lipophilic balance (HLB value) and other critical parameters are discussed. It is believed that this approach may very well be essential for advancing the applicability of these water-based, biocompatible, and often highly dichroic materials for a wide range of uses.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.201701209. many new and exciting applications are emerging that require novel materials properties and new engineering methods. The last decade has seen an increasing interest in synergizing unique LC characteristics with aqueous environments, for instance, for the development of biological sensing applications, [1][2][3][4][5][6][7] (biocompatible) templates which align and manipulate (living) anisotropic materials, [8][9][10][11][12][13] and highly dichroic LCs for facile fabrication of optical elements. [14][15][16][17][18] This particular field of application requires different, water compatible materials. Excellent candidates are so-called lyotropic chromonic liquid crystals (LCLCs), which are rigid, plank-like molecules consisting of aromatic cores with hydrophilic groups on the periphery that, in water, spontaneously stack face-to-face in columnar assemblies due to π-π stacking and hydrophobic interactions. At certain concentrations and temperatures, these stacks form liquid crystalline phases [14,15] such as nematic and columnar phases. Typical members of the class of LCLCs are highly conjugated dyes that are functionalized with water-soluble (often ionic) groups. [14] The rigid, aromatic cores of LCLCs give these materials excellent optical properties, such as strong linear dichroism, high birefringence, and in some cases high absorbance in the visible range.Typically, LCLCs are applied either in closed cells where one benefits from the dynamics of the anisotropic solution or, alternatively, as a thin film after water evaporation from an anisotropic LCLC solution. In any application, however, high-quality, homogeneous, and stable macroscopic alignment is ...