Nanoparticles (NPs) are essential in nanomedicine for imaging and therapy. When introduced into the body, they are protected from the immune system by polyethylene glycol (PEG). PEGylation strategies influence NP size, shape, density, loading, molecular weight, charge, and purification. Incorporating targeting ligands is also common. This article provides an overview for newcomers to stealth NPs, highlighting key considerations for PEGylation protocols and performance characterization. NPs, ranging from 1 to hundreds of nanometers, have unique interactions with matter, enabling applications in biomedicine. Over 35 FDA-approved NPs incorporate PEG, with many in preclinical studies for imaging and therapy. NPs have large payloads, stability, avidity, and signal enhancement due to their size and surface area-to-volume ratio. They behave differently from other therapies and imaging agents, affecting in vivo applications. For example, NPs extravasate more in cancer tissue and remain due to the enhanced permeability and retention (EPR) effect. Despite advantages, challenges like RES uptake and nonspecific binding hinder NP deployment. PEGylation reduces RES uptake and increases circulation time. PEG chains reduce charge-based interactions, increase solubility, and modulate the EPR effect. PEGylated NPs accumulate less in the liver and more in tumors compared to non-PEGylated NPs. PEG is inexpensive, versatile, and FDA-approved. PEGylation affects NP behavior, including circulation time, aggregation, and interactions with the RES. Larger PEG chains increase circulation time but may reduce tumor accumulation if too large. PEG length and conformation influence NP behavior, with brush-like PEG configurations providing better stealth. PEG termini affect in vivo behavior, with certain termini reducing nonspecific binding. PEGylation methods include covalent and noncovalent approaches. Covalent methods use thiol or carboxyl groups, while noncovalent methods use lipid-PEG conjugates. Characterization methods like dynamic light scattering measure NP size, zeta potential, and size distribution. PEG loading levels are determined by various metrics, including PEG:NP ratio and PEG footprint. Ligands enhance NP targeting by increasing tumor accumulation and site-specific accumulation. PEGylation with reactive termini allows for ligand binding. Alternatives to PEG include natural products, PEG hybrids, and next-generation polymers. Challenges include PEG toxicity, degradation by light, heat, or shear stress, and the need for biodegradable coatings. In conclusion, PEGylation is crucial for NP stability and in vivo performance. Future research aims to improve NP targeting and delivery, with strategies combining signaling and therapeutic benefits. PEG remains a key component in nanomedicine, though challenges persist in its application.Nanoparticles (NPs) are essential in nanomedicine for imaging and therapy. When introduced into the body, they are protected from the immune system by polyethylene glycol (PEG). PEGylation strategies influence NP size, shape, density, loading, molecular weight, charge, and purification. Incorporating targeting ligands is also common. This article provides an overview for newcomers to stealth NPs, highlighting key considerations for PEGylation protocols and performance characterization. NPs, ranging from 1 to hundreds of nanometers, have unique interactions with matter, enabling applications in biomedicine. Over 35 FDA-approved NPs incorporate PEG, with many in preclinical studies for imaging and therapy. NPs have large payloads, stability, avidity, and signal enhancement due to their size and surface area-to-volume ratio. They behave differently from other therapies and imaging agents, affecting in vivo applications. For example, NPs extravasate more in cancer tissue and remain due to the enhanced permeability and retention (EPR) effect. Despite advantages, challenges like RES uptake and nonspecific binding hinder NP deployment. PEGylation reduces RES uptake and increases circulation time. PEG chains reduce charge-based interactions, increase solubility, and modulate the EPR effect. PEGylated NPs accumulate less in the liver and more in tumors compared to non-PEGylated NPs. PEG is inexpensive, versatile, and FDA-approved. PEGylation affects NP behavior, including circulation time, aggregation, and interactions with the RES. Larger PEG chains increase circulation time but may reduce tumor accumulation if too large. PEG length and conformation influence NP behavior, with brush-like PEG configurations providing better stealth. PEG termini affect in vivo behavior, with certain termini reducing nonspecific binding. PEGylation methods include covalent and noncovalent approaches. Covalent methods use thiol or carboxyl groups, while noncovalent methods use lipid-PEG conjugates. Characterization methods like dynamic light scattering measure NP size, zeta potential, and size distribution. PEG loading levels are determined by various metrics, including PEG:NP ratio and PEG footprint. Ligands enhance NP targeting by increasing tumor accumulation and site-specific accumulation. PEGylation with reactive termini allows for ligand binding. Alternatives to PEG include natural products, PEG hybrids, and next-generation polymers. Challenges include PEG toxicity, degradation by light, heat, or shear stress, and the need for biodegradable coatings. In conclusion, PEGylation is crucial for NP stability and in vivo performance. Future research aims to improve NP targeting and delivery, with strategies combining signaling and therapeutic benefits. PEG remains a key component in nanomedicine, though challenges persist in its application.