<p>State transition (ST) is a key regulatory mechanism that balances excitation energy between photosystem II (PSII) and photosystem I (PSI). Although its general principles have been known for decades, methodological constraints have limited precise quantification of ST kinetics and of the associated changes in photosystem absorption cross sections. We present a PCA‑assisted fluorescence spectral analysis that enables in vivo separation of PSI and PSII chlorophyll fluorescence at physiological temperatures. Applied to intact <i>Nicotiana tabacum</i> leaves undergoing ST, this method provides more accurate estimates of both the rates and the extent of the process than earlier approaches. The half‑time of the State 1 to State 2 (St1→St2) transition was approximately 1 min, nearly twice as fast as values inferred from traditional F_far‑red/F_red fluorescence ratios measured at room temperature or 77 K. The analysis also clarifies how PSI and PSII fluorescence changes relate to variations in their absorption cross sections. Accounting for PSII‑to‑PSI spillover, we found that the mobile LHCII fraction associating with PSI during the St1→St2 transition increased the PSI absorption cross section by 31%, whereas LHCII reassociation with PSII during the reverse transition produced a more modest 17% increase in PSII absorption cross section. These asymmetric adjustments likely have important physiological implications under fluctuating environmental conditions. Finally, applying the method to isoprene‑emitting and non‑emitting tobacco lines revealed that isoprene accelerates excitation‑energy redistribution during the St2→St1 transition, consistent with its proposed role in maintaining optimal thylakoid ultrastructure and fluidity. Overall, this approach offers a powerful tool for dissecting photosynthetic energy‑distribution dynamics in vivo.</p>