Lateral flow immunoassay (LFIA) is a widely used point-of-care method for both qualitative and semi- or fully quantitative detection of clinical biomarkers. There has, however, been limited data available on direct comparison of different types of labels in the LFIA for quantitative analysis using the same non-competitive assay setup. In this study, two groups of antibody-conjugated nanoparticle labels are compared: conventional gold nanostructures with densitometry readout and upconversion nanoparticles (UCNPs) suitable for sensitive luminescence readout. First, the LFIA procedure was optimized using human serum albumin (HSA) as an analyte. In the group of gold-based labels, standard gold nanoparticles were compared with gold nanoshells (AuNSs). In the case of UCNP labels, doping with Er³⁺ and Tm³⁺ and surface modifications based on poly(ethylene glycol) and poly(acrylic acid) (PAA) were compared. The best representatives—AuNSs and PAA-Tm UCNPs—were successfully used to detect human serum albumin (HSA) in spiked urine samples. To assess assay versatility, prostate-specific antigen (PSA) was detected in serum. While AuNSs tended to aggregate in serum, the PAA-Tm UCNPs maintained their stability and achieved the limit of detection of 34 pg/mL. Finally, spiked serum samples were analyzed to evaluate detection trueness, achieving a strong correlation with the reference concentrations (recoveries ranging from 82 to 118%). We showed that both label choice and sample matrix are critical: while AuNSs and PAA-Tm UCNPs performed well in urine, PAA-Tm UCNPs excelled in the more complex serum matrix. Thus, PAA-Tm UCNPs were found to be the most suitable labels for biomarker quantitation, highlighting the strong potential of UCNPs for sensitive LFIA applications.