Spinel, an important mineral in basalts and ultramafic rocks on Earth, Mars, and the Moon, is sensitive to petrologic and geochemical processes, and redox evolution. Due to the small grain size of extraterrestrial samples, investigations on the composition of spinel samples including presence of trace elements and ferric iron have been hindered by the lack of appropriate in-situ analytical techniques with high spatial resolution and the shortage of reference materials. This paper presents a combined method of simultaneously measuring the major and trace elements, and Fe3+/∑Fe ratio in spinel samples using electron probe microanalysis (EPMA). Our new EPMA method is performed under double beam condition at a beam current of 200 nA for trace elements (Ti, V, Mn, Co, Ni, and Zn) and 60 nA for major elements (Mg, Fe, Al, and Cr) with an acceleration voltage of 25 kV. In addition, large analyzing crystals and peak overlap corrections were applied to reduce the detection limits and improve the analytical precision. The detection limits of 16–55 μg/g (3σ) for trace elements were achieved, and the estimated accuracies for the major elements and trace elements were within ± 2 and ± 6% (1σ), respectively. We selected seven spinel samples from the Luobusha and Stillwater intrusions to evaluate the validity of our method. They were sufficiently homogeneous with a relative standard deviation (RSD) of ± 2.0% (1σ) for the major elements (except MgO in 16SW3-9) and ± 7.0% (1σ) for the trace elements. The EPMA results obtained for the major and trace elements of the most homogeneous spinel LBS13-04 were compared with those measured using X-ray fluorescence and laser ablation inductively coupled plasma mass spectrometry. These values were in good agreement with the uncertainty of the methods. Thus, this spinel is highly suitable as a reference material for in situ microanalysis. The Fe3+/ΣFe ratios of high Cr# (57.7-79.1) spinel standards determined using M?ssbauer spectroscopy varied from 0.07 to 0.27, which were used for secondary standard calibration method to determine the spinel Fe3+/ΣFe ratio with an accuracy of < ± 0.04 (2σ). Our results offer a high-precision EPMA method that can simultaneously determine the major and trace elements together with the Fe3+/ΣFe ratio in spinel. This method provides robust and precise data on spinel for small, precious, and rare terrestrial or extraterrestrial samples, which can be used to understand the formation and evolution of rocky planets.