Veterinary laboratories face distinct challenges in Pakistan, including inadequate infrastructure, resources, and training opportunities, especially in the Khyber Pakhtunkhwa and Balochistan regions. This study aimed to evaluate the impact of training sessions for veterinary laboratory staff to improve methods and protocols related to sample collection, storage, and transport, while ensuring strict compliance with biosafety and biosecurity guidelines. The study employed a mixed methods approach, incorporating qualitative and quantitative research techniques. Hands-on training, essential laboratory equipment, and a comprehensive training kit, including personal protective equipment (PPE), were provided to 13 laboratories within the Livestock and Dairy Development Departments of Khyber Pakhtunkhwa and Balochistan. A random sample of 152 individuals from a cohort of 314 trained personnel was selected to assess procedural changes post-training, supplemented by Training Needs Assessments (TNAs) and follow-up visits. Data collection involved a combination of open- and closed-ended questionnaires, individual interviews, and focus group discussions by trained enumerators to maintain a standardized approach. Significant improvements were observed in laboratory practices and procedures, staff competency in sample collection, necropsy techniques, labeling, storage, a chain of custody, packaging, and transport, as well as biosafety and biosecurity practices, such as effective use of PPEs, good laboratory practices, standard operating procedures, handling of sharps, and waste management. However, areas needing refinement, particularly waste management protocols, were identified. The integrated approach combining TNAs, training initiatives, and resource distribution, including laboratory equipment and PPEs, was pivotal in achieving these outcomes. This comprehensive strategy provides a basis for improving biosafety and biosecurity measures within laboratories, thereby contributing to the global effort to mitigate unauthorized access to high-risk pathogens.