Precise visualization of nanoparticles (NPs) at the subcellular level is crucial for targeted drug delivery. However, NPs functionalized with permanently fluorescent probes are challenging to detect when only a small fraction reaches the target compartment, as weak fluorescence signals are obscured by intense fluorescence from NPs accumulated in off-target subcellular compartments. Therefore, the aim of this work was to establish fluorogenic NPs that activate their fluorescence only at the intracellular target upon the inverse electron demand Diels–Alder (iEDDA) click reaction. The fluorogenic moiety on the NP surface was a coumarin-102–tetrazine (C102-Tet), while trans-cyclooctene (TCO) served as the second reaction partner immobilized on microtubules using anchors of different geometries and spatial accessibilities. We generated polymer NPs composed of poly(lactide-co-glycolide) (PLGA) and poly(ethylene glycol)-poly(lactic acid) (PEG–PLA) by systematically varying the PEG chain length (2k vs 5k) and C102-Tet surface density. Direct stochastic optical reconstruction microscopy (dSTORM) resolved individual NPs of ∼80 nm and confirmed that C102-Tet presented on the NP surface remains reactive. Quantitatively, 15–45% of the C102-Tet groups participated in the iEDDA reaction, depending on PEG chain length and surface density. To overcome insufficient endosomal escape, which is a still-unsolved challenge for nano-sized drug delivery systems, NPs were directly delivered into the cytosol to prove the fluorogenic NP concept that becomes activated only upon bio-orthogonal reaction at the intracellular target. Confocal microscopy and super-resolution nonlinear structured illumination microscopy imaging revealed NP fluorescence aligned along microtubules and supported target-specific iEDDA activation on the surface of NPs. This strategy enables high-contrast tracking of nanocarriers at defined intracellular targets.