The periodontal ligament (PDL) is crucial for maintaining the integrity and functionality of tooth-supporting structures. Mechanical forces applied to the tooth during orthodontic tooth movement generate pore pressure gradients, leading to interstitial fluid movement within the PDL. The generated fluid shear stress (FSS) stimulates the remodeling of PDL and alveolar bone. Herein, we present the construction of a parallel fluid-flow apparatus to determine the effect of FSS on PDL cells. The chamber was designed and optimized using computer-aided and computational fluid dynamics software. The chamber was formed by PDMS using a negative molding technique. hPDLCs from two donors were seeded on microscopic slides and exposed to FSS of 6 dyn/cm² for 1 h. The effect of FSS on gene and protein expression was determined using RT-qPCR and Western blot. FSS upregulated genes responsible for mechanosensing (FOS), tissue formation (RUNX2, VEGFA), and inflammation (PTGS2/COX2, CXCL8/IL8, IL6) in both donors, with donor 2 showing higher gene upregulation. Protein expression of PTGS2/COX2 was higher in donor 2 but not in donor 1. RUNX2 protein was not expressed in either donor after FSS. In summary, FSS is crucial in regulating gene expression linked to PDL remodeling and inflammation, with donor variability potentially affecting outcomes.