Acoustic levitators are devices which generate converging acoustic radiation forces and thus can trap objects in mid-air. Acoustic levitators have found applications in the fields of chemistry and biology as a non-contact transportation method. The trapping of objects can be achieved using a phased-array in which the phase of the signal sent to each transducer is varied to generate a trap that stably holds the particle at the target three-dimensional position. The transducer phases can be changed over time to translate the acoustic field in space, thereby transporting the trapped particle. Here, we describe an open-loop spatial calibration scheme which increases the positioning accuracy of a particle in an acoustic levitator. The effectiveness and the performance of the spatial calibration was determined using a single-axis standing wave levitator with 60 ultrasonic transducers (40 kHz), and a levitated particle (EPS particle of radius 0.7 mm). Our calibration method is shown to significantly improve the positioning accuracy of the particle inside the acoustic levitator and reduced RMS error down to 0.11 and 0.03 mm in x and z axes, respectively. Although the calibration approach only considers the static response, the trajectory when the particle moves at a relatively high velocity (≈1 cm/s) was also improved. Increasing the precision and velocity of a moving particle will enhance the capabilities and reliability of acoustic levitators and open up possibilities for novel applications.