Reference conditions having no or only minor anthropogenic disturbances, are a basic requirement for ecological studies in rivers. To study ecological impact of a dam, two sites were pre-classified as reference or least disturbed in the Andhi Khola River, Nepal, using Rapid Field Bioscreening (RFB) protocol. Biological (macroinvertebrates) and physico-chemical samples were collected in the month of January and February 2013. Multi-habitat sampling (MHS) was employed in sample collection of macro-invertebrates fauna. Validation of the pre-classified sites was done using several indices viz. Nepalese biotic score (NEPBIOS), Biological monitoring working party (BMWP), Hindu-Kush Himalayan biotic score (HKHBIOS), Hilsenhoff (HILSENHOFF) & National Sanitation Foundation Water Quality Index (NSFWQI). The NEPBIOS, HKHBIOS, HILSENHOFF, RFB & NSFWQI indices predicted the river quality at all two sites as good with quality class II. Only BMWP/ASPT water quality index predicted the river quality at all two sites as Excellent with quality class I. Hence preselected sites with quality of rank II i.e. good qualities were validated as reference sites. The study demonstrated that the multi-metric approach is suitable for application in the monitoring and assessment of rivers where dams are built to produce hydropower.