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Gastro-retentive drug delivery systems (GRDDS) like gastro-retentive microspheres have gained immense popularity in the field of oral drug delivery. It is a widely employed approach to retain the dosage form in the stomach for an extended period of time and release the drug slowly that can address many challenges associated with conventional oral delivery, including poor bioavailability. Different innovative approaches like magnetic field assisted gastro-retention, swellingsystems, mucoadhesion techniques, floating systems with or without effervescence are being applied to fabricate gastroretentive microspheres. Apart from in-vitro characterization, successful gastro-retentive microspheres development demands well designed in-vivo study to establish enhanced gastro-retention and prolonged drug release. Gama scintigraphy and MRI are popular techniques to evaluate in-vivo gastric residence time. However, checking of their overall in-vivo efficacy still remains a major challenge for this kind of dosage form, especially in small animals like mice or rat. Reported in-vivo studies with beagle dogs, rabbits, and human subjects are only a handful in spite of a large number of encouraging in-vitro results. In spite of the many advantages, high subject variations in gastrointestinal physiological condition, effect of food, and variable rate of gastric emptying time are the challenges that limit the availability of gastro-retentive microspheres in the market.