A method for evaluation of gastric emptying via monitoring serial serum levels of acetaminophen (APAP) as an indicator, without involving animal sacrifice, has been developed in conscious rats. We administered orally suspended test meals of 0.5, 1, 2, or 4 mL/rat each, containing 20 mg APAP. The animals were repeatedly bleed at 15, 30, 45, 60, or 90 min after APAP treatment. Serum APAP level was determined by a high-performance liquid chromatography. As a result, a 2-mL meal was found optimal to attain continuous levels of APAP in serum. The APAP method showed a close correlation (r = 0.77) with the phenol red method, a standard procedure for quantitative detection of gastric emptying entailing rat sacrifice. The blood loss (approximately 2 mL) resulting from the APAP method did not significantly modify the rate of gastric emptying. In addition, data was presented to substantiate the validity of the APAP method in demonstrating enhancement and inhibition of gastric emptying using cisapride and cholecystokinin-octapeptide (CCK), respectively. These results suggest that the APAP method is easy, versatile, and reliable, and requires only a minimal number of animals for the evaluation of sequential changes in gastric emptying.