The zero-field splitting parameters (ZFS) of Mn4+ and Fe3+ ions in LiAlO2 with a layered structure are analyzed experimentally and theoretically by using high-frequency electron paramagnetic resonance spectroscopy, Neuman superposition model (NSM), DFT and multiconfigurational calculations. The interpretation of ZFS is based on the comparison of the experimentally determined values with the calculated ones. This approach allows assessing the performance of different methods for computation of ZFS of Fe3+ and Mn4+ in layered oxide matrices. DFT and multiconfigurational calculations are used to analyze the effect of oxygen, aluminium, and lithium neighbours on ZFS of Fe3+ and Mn4+. These calculations are based on a cluster comprising Fe3+ or Mn4+ ions in a trigonally compressed octahedron with 6 metal ions (Al3+ or Co3+) as first metal neighbours and 6 O2− and 2 Li+ (above and below the layer) as second neighbours. A satisfactory agreement with the experimental data is achieved when the local structure of Mn4+ and Fe3+ deviates from the trigonal host-site geometry. The local structure of Fe3+ comprises an axial distortion, while trigonal environment with reduced extent of distortion appears around Mn4+.