Cystinuria is a rare congenital disorder characterized by the formation of cystine stones in urinary system, mainly kidneys and urinary tract. It follows the autosomal recessive inheritance pattern, where both of the parents contain the mutant allele. Cystine is an oxidized dimeric form of amino acid cysteine, shining crystal of greenish-yellow color sized greater than 5 mm. A minor genetic defect in SLC3A1 gene, downregulate the cystine transporter, rBAT protein, to absorb cystine and other dibasic amino acids in proximal tubule of nephron, causing Cystinuria. Computational and molecular analysis of SLC3A1 gene was performed to identify the deleterious missense single nucleotide variations (mSNVs) linked with Cystinuria in Pakistani population. In silico analysis of whole SLC3A1 gene nsSNPs has revealed that the exon 1, 6 and 10 are the hotspot areas, which potentially alter the protein structure and function. Three SNVs including one synonymous SNV A186C in exon 1, and two mSNVs including G314T in exon 1 and G44972A in exons 10 were identified. Both mSNVs were confirmed by ARMS PCR in all the 68 samples. The results have shown that 10% of the patients have G314T, 16% have G44972A and 74% of the patients have both of these mSNVs. Both of these mSNVs were involved in the structural and functional deterioration of rBAT protein. Additionally, computer aided drug designing tools were used to design diaminobenzylpyrimidine drug around the mutant residues which exhibit the lowest binding affinity with the target as compared to the previously reported cystine binding thiol drugs. In future, the present study could be extended to a large scale for mass screening of reported SNVs and mSNVs which, ultimately, lead to the development of knockouts for the functional studies and treatments.