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Radio Frequency Identification (RFID) system is a communication technology used to identify objects using electromagnetic waves. The key advantage of RFID systems stems from their ability to simultaneously identify multiple tagged objects. However, communication of multiple tags with a reader may result in a collision problem, which is both time and energy inefficient, hindering the effectiveness of tag identification process. Presently, several anti-collision algorithms can be applied in order to reduce the collision probability. The reader¿s a priori knowledge of tag quantity significantly affects the overall performance of the system. Since the exact number of tags is not available for the reader, it is essential to develop an accurate tag estimation method to increase the efficiency of tag identification process. This paper presents a novel tag quantity estimation method, whereby, after simulating the tag distribution process, cubic spline interpolation method is employed to approximate the number of tags. According to the simulation results and the evaluation of the previous estimation methods, the new proposed method estimates the number of tags with a higher accuracy yielding an error rate of less than 1%, on average. Moreover, this low error rate is preserved even when the number of tags increases considerably.