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The quality and freshness of aquatic products are critical parameters for both consumers and stakeholders in the fisheries sector. Spectral techniques have demonstrated strong potential in assessing not only the freshness status but also the authenticity (e.g., frozen vs. thawed) of various fish species. Recent findings indicate that mitochondria can serve as reliable biomarkers for evaluating the freshness of aquatic products and distinguishing between fresh and thawed fillets based on their post-mortem oxidative state. This study evaluates the potential and complementarity of several spectral methods – including infrared spectroscopy, fluorescence spectroscopy, and low-field nuclear magnetic resonance (NMR) – for determining freshness and authenticity in two commercially important fish species: seabass and gilthead seabream. Analyses were conducted on both fillets and isolated mitochondria. The results highlight that isolated mitochondria may serve as effective traceability markers, providing a promising tool for quality monitoring of fresh and frozen fillets using non-destructive spectral techniques.