A rapid SE-HPLC-ICPMS method was developed to analyze REE-UCNPs (NaYF4:Yb3+, Tm3+) and their degradation products in complex matrices. Its application to rat serum revealed Y3+ and Yb3+ release profiles during nanoparticle degradation. This study offers key insights into the biological quantitative analysis of UCNPs, facilitating further research on their biodistribution and toxicology.