**Background:** The international scientific community has recognized fish as sentient beings, prompting countries to implement regulations for farmed fish welfare. However, existing guidelines focus mainly on transport and slaughter, neglecting species-specific needs and critical welfare points throughout the entire production cycle. Nile tilapia (Oreochromis niloticus) is among the three most farmed fish globally, with major producers including China, Indonesia, Egypt, Bangladesh, and Brazil. The aquaculture sector faces pressure to increase production with fewer resources, which can negatively impact fish welfare. Previous protocols for tilapia welfare were limited to the grow-out phase. This study aimed to develop a comprehensive welfare assessment protocol covering all life stages—reproduction, nursery, and grow-out—and to establish quantitative welfare indices.
**Methods:** The study employed an integrative systematic literature review (1985–2023) using Google Scholar to define welfare indicators and reference values for each cultivation stage. Indicators were organized according to four of the five animal freedoms: environmental, health, nutritional, and behavioral. Reference values were categorized into three scores: 1 (ideal), 2 (tolerable), and 3 (critical). A laboratory experiment was conducted at the Federal University of Paraná, Brazil, using 480 newly hatched tilapia larvae subdivided into 12 aquariums under controlled conditions (temperature 27.01 ± 1.0°C, pH 7.83 ± 0.2, dissolved oxygen 5.40 ± 0.4 mg/L). Over 15 days, one larva per aquarium was randomly selected daily, anesthetized with clove oil (500 mg/L), and evaluated under a stereo microscope for health indicators (eyes, mouth/jaws, skin, fins, gill covers, spine, yolk sac). Partial welfare indices (PWIs) were calculated for each freedom using weights based on the number of scientific publications (ln of document count), and a general welfare index (GWI) was derived as the arithmetic mean of PWIs multiplied by an elimination factor (kl). The elimination factor is set to 0 if mortality exceeds 30%, automatically classifying welfare as critical.
**Key Results:** The final protocol includes 12 environmental indicators (e.g., temperature, pH, dissolved oxygen, non-ionized ammonia, nitrite, alkalinity, photoperiod, transparency, predators, hapa net cleaning, sex ratio), with reference values specific to each phase. For example, ideal temperature range is 24.0–31.0°C (score 1), tolerable 21.0–23.9 or 31.1–34.9°C (score 2), and critical ≤20.9 or ≥35.0°C (score 3). Health indicators for breeders and grow-out include eyes, jaw/lips, operculum, skin, fins, gills, spine, mortality (ideal ≤10%, score 1; 11–24% score 2; ≥25% score 3), and invasive procedures. For nursery phase, indicators are subdivided by developmental stage (larvae, post-larvae, fingerlings) and include hatching rate (ideal ≥90%), eyes, jaws/lips/head, skin, tail, spine, yolk sac, operculum, fins, emaciation state, and mortality (ideal ≤10%). Nutritional indicators include feed crude protein, amount of feed, feeding frequency, and food distribution, with values adjusted by fish weight. Behavioral indicators include anesthesia effectiveness, feed intake (ideal 180–300 minutes), swimming behavior, and stunning reflexes during slaughter. The weights for indicators ranged from 2 to 10, based on publication counts. A simulated example for the grow-out phase yielded a GWI of 0.59, classified as moderate, with high confidence.
**Clinical Implications:** This protocol provides a standardized, quantitative tool for tilapia farmers to monitor and improve welfare across all production stages, from reproduction to slaughter. The use of simple, non-invasive, low-cost indicators makes it practical for commercial operations. The indices allow for objective comparison between farms, locations, and time periods, enabling identification of areas needing improvement. The approach supports the adoption of more sustainable and ethical practices, potentially enhancing product quality and consumer trust. The study also highlights the importance of including early life stages in welfare assessments, based on the precautionary principle regarding larval sentience. The protocol can be updated as scientific knowledge advances, and its application may help meet growing market demands for certified animal welfare in aquaculture.