Abstract: Histone H3 clipping, a unique but evolutionarily conserved post-translational modification that irreversibly removes the N-terminal tail of H3, has been reported across diverse eukaryotic lineages. In Tetrahymena thermophila, a ciliate with nuclear dimorphism, H3 clipping is a bona fide proteolytic event generating H3^F (H3-Fast) by removing the first six N-terminal amino acids, specifically in the transcriptionally silent micronucleus (MIC). However, the detection of H3^F remains technically demanding, time-consuming, and lacks spatio-temporal resolution. To overcome this, a 2 × branched peptide antigen was developed to generate a high-specificity antibody that exclusively recognizes H3^F, effectively distinguishing it from full-length H3 and other truncation variants. This antibody eliminated the need for labor-intensive MIC isolation and histone extraction, enabling rapid, small-scale detection directly from whole-cell lysates. Using this antibody, dynamic subcellular localization of H3^F was investigated through different cell stages, revealing its persistence during vegetation, starvation and early conjugation. However, H3^F disappeared concurrently with macronuclear anlage formation, supporting the notation that removal of H3^F is a prerequisite for the new macronucleus development. Comparative analyses further revealed that H3 Ser10 phosphorylation, though previously used as an alternative H3^F marker, actually occurs strictly after clipping, refining the temporal hierarchy of these two chromatin events. This work provides the first in situ, high-resolution method to track endogenous H3 clipping, providing both a technical platform and new biological insight into the developmental regulation of proteolytic histone modifications.