Background/Objectives: Nanoparticles have emerged as indispensable tools in modern biomedicine, enabling precise diagnostics, targeted therapy, and controlled drug delivery. Despite their rapid progress, the translation of nanoparticle-based systems critically depends on the ability to detect, quantify, and track them across complex biological environments. Over the past two decades, a wide spectrum of detection modalities has been developed, encompassing optical, magnetic, acoustic, nuclear, cytometric, and mass spectrometric principles. Yet, no comprehensive framework has been established to compare these methods in terms of sensitivity, spatial resolution, and clinical applicability. Methods: Here we show a systematic analysis of all broadly applicable nanoparticle detection strategies, outlining their mechanisms, advantages, and drawbacks, and providing illustrative examples of practical applications. Results: This comparison reveals that each modality occupies a distinct niche: optical methods offer high sensitivity but limited penetration depth; magnetic and acoustic modalities enable repeated non-invasive tracking; nuclear imaging ensures quantitative, whole-body visualization; and invasive biochemical or histological assays achieve ultimate detection limits at the cost of tissue integrity. These findings redefine how each technique contributes to nanoparticle biodistribution and mechanistic studies, clarifying which are best suited for translational and clinical use. Conclusions: Placed in a broader context, this review bridges fundamental nanotechnology with biomedical applications, outlining a unified methodological framework that will guide the rational design, validation, and clinical implementation of nanoparticle-based therapeutics and diagnostics. By synthesizing the field into a single comparative framework, it also provides an accessible entry point for newcomers in nanotechnology and related biomedical sciences.