Alzheimer's disease (AD) is closely associated with cholinergic dysfunction, and dual inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) represents a promising therapeutic strategy. However, current screening methods for cholinesterase inhibitors, particularly Ellman-based assays, are prone to spectral interferences and limited analytical specificity, which may compromise accurate identification and kinetic characterization of potent inhibitors. In addition, highly potent dual AChE/BuChE inhibitors with well-defined structure-activity relationships remain limited. To address these limitations, this study combines rational molecular design with the development of a selective chromatographic screening platform based on magnetic particle-immobilized enzymes and post-column derivatization, enabling improved analytical specificity and reliable quantification of enzymatic activity. In this work, a series of novel coumarin-benzothiazole hybrids were designed, synthesized, and evaluated as potential dual ChE inhibitors. The target compounds were obtained through a multistep synthetic strategy involving formation of coumarin-benzothiazole 10 (90% yield), O-alkylation (81-89% yield), and nucleophilic substitution with nitrogen heterocycles to afford hybrids 13a-p in yields ranging from 19 to 92%. The compounds were first screened using a newly developed semi-automated chromatographic platform, which combines magnetic particle-immobilized enzymes with post-column derivatization, overcoming limitations of conventional Ellman-based assays. Donepezil was used as a proof-of-concept reference inhibitor, confirming the accuracy of the method. The analytical method demonstrated excellent linearity (R2 = 0.9983-0.9992), precision (CV ≤ 7.8%), and accuracy within accepted validation limits. Biological evaluation revealed highly potent inhibitors, particularly 13a and 13m, which exhibited IC₅₀ values of 1.0 ± 0.1 nM and 0.6 ± 0.1 nM against AChE, respectively, compared with 64.9 ± 16.0 nM for donepezil. For BuChE, 13m showed an IC₅₀ of 109.2 ± 14.7 nM, while 13a displayed 1212.5 ± 144.0 nM, both lower than donepezil (2202 ± 326 nM). Kinetic studies confirmed a competitive mechanism of inhibition, with Kᵢ values as low as 0.12 ± 0.02 nM for AChE and 47.76 ± 15.14 nM for BuChE (13m). Molecular docking studies supported these findings, demonstrating key interactions within the catalytic sites of both enzymes. Additionally, in silico evaluations indicated that these derivatives have favorable druglikeness. Together, the results highlight coumarin-benzothiazole derivatives as promising dual-target ChEIs and establish the developed analytical method as a versatile tool for the discovery and characterization of new anti-AD candidates.