Best practices for designing a multi-tier architecture in a VPC include separating web, application, and database tiers into different subnets, keeping application and database servers in private subnets, deploying resources across multiple Availability Zones, using load balancers and Auto Scaling for scalability, implementing least-privilege Security Groups, using NAT Gateways and VPC Endpoints for secure connectivity, and enabling VPC Flow Logs and CloudWatch for monitoring. This design improves security, availability, and fault tolerance.