The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca²⁺signals with Ca²⁺reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca²⁺transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca²⁺signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca²⁺channel Orai1 and Gα_i-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca²⁺signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca²⁺oscillations. Together, these findings establish Ca²⁺frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.