Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cell contraction. α₂-Adrenoceptor agonists and antagonists may have important applications in cardiovascular drug development. To assess α₂-adrenoceptor-mediated drug effects on vascular smooth muscle contraction, we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation (pMLC₂₀) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human α_2B-adrenoceptor (A7r5-α_2B cell line). Methods: In a 96-well format, confluent and serum-starved cells (+/− inhibitor preincubation) were treated with receptor ligands for 5–120 s and the evoked pMLC₂₀ response was monitored with a quantitative in-cell immunoassay, employing time-resolved fluorescence technology. Western blotting, immunofluorescent labelling and intracellular calcium concentration measurements were used for assay validation. Results: The α₂-adrenoceptor agonist dexmedetomidine induced rapid, transient and dose-dependent (EC₅₀ 30–65 nM) myosin light chain phosphorylation, peaking at 20–45 s with an E_max value of approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responses that were comparable to those evoked by dexmedetomidine. Blockers of α₂-adrenoceptors, myosin light chain kinase, G_i-proteins, Gβγ subunits, L-type calcium channels and phospholipase C antagonized the dexmedetomidine-evoked myosin light chain phosphorylation, whereas blockers of protein kinase C and protein kinase A potentiated the response to dexmedetomidine. Discussion: The novel method is suitable as a ligand profiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction and for investigating the intracellular pathways involved in this process. The assay now allows the quantitative determination of pMLC₂₀ signal induction or inhibition in vascular smooth muscle cells and is superior to conventional Western blotting due to the reduced number of cells required and the potential for measurement of detailed time curves, multiple treatments and replicates on each plate.