Background: This 5-year external quality assessment (EQA) study evaluated laboratory performance in analyzing seminal plasma biomarkers (zinc, citrate, fructose, neutral α-glucosidase (NAG)), assessing inter-lab variability, methodological trends, and technique-related differences. Methods: From 2021 to 2025, a total of 113 laboratories participated in this EQA program. Standardized lyophilized seminal plasma samples were prepared from pooled ejaculates and adjusted to target biomarker concentrations. Participants analyzed these samples using their routine methods. Results: Over the five-year period, zinc exhibited the lowest mean coefficient of variation (CV) at 27.0%(range: 15.1–50.5%), with the Five-Br-2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (Five-Br-PAPS) method demonstrating superior precision (mean CV: 14.5%) compared to the resorcinol monosodium salt (PAR) (21.5%) and 1-(2-Pyridylazo)-2-naphthol (PAN) (19.9%) methods, though inter-method differences were not statistically significant. For citrate, the mean CV was 41.9% (range: 16.6–62.7%), with no significant difference between the citrate lyase (mean CV: 36.8%) and Fe3+ complexation methods (36.3%). Fructose analysis showed the highest variability, with a mean CV of 44.7% (range: 22.7–63.9%). The hexokinase method (mean CV: 39.5%) outperformed the indole method (49.5%), though the difference was not statistically significant (p = 0.213). NAG exhibited a mean CV of 42.0% (range: 25.1–59.1%), with the kinetic method (mean CV: 25.7%) showing significantly better precision than the endpoint method (44.2%). Conclusions: The study highlights inter-laboratory variability in seminal plasma biochemical testing, underscoring the need for standardized methodologies and enhanced quality control. Continuous EQA participation and targeted training are essential to improve diagnostic reliability in male infertility assessments.