The "catalytic triad" present at the active site of ribonuclease A (RNase A) is responsible for the cleavage of the 5'-phosphodiester bond; amino acid residues His12, Lys41 and His119 constituting this triad provide a positively charged environment at the physiological pH. Based on docking studies, 1,4,5-trisubstituted-carboxylated 1,2,3-triazoles (1,4,5-TTs) were identified as a new class of RNase A inhibitors. Therefore, two different groups of 1,4,5-TTs, functionalized with carboxylic acid groups, were synthesized by reacting pre functionalized butyne-1,4-diol derivatives with several aryl/alkyl azides under solvent and catalyst free conditions. Inhibitory properties of the new molecules with heteroatom linked carboxylic acid "CH2XCH2CO2H" (X = S, O) functionalities were investigated by performing qualitative and quantitative biophysical studies. All the "CH2S" and "CH2O" linked acid derivatives (6a-e, 6f'-g' and 6h, and 8a-e, 8f'-g' and 8h) exhibited significant competitive inhibition with inhibition constant values (Ki) ranging from 9 to 34 μM determined by steady state enzyme kinetics. Uracil based bisthioglycolic acid (6h) and carboxylic acid based bisoxyacetic acid (8g') derivatives were found to be the most promising inhibitors with Ki values of 9.9 ± 0.7 and 15.6 ± 0.6 μM, respectively. Additional molecular docking studies revealed that a sufficient number of hydrogen bonding interactions were generated from various functional groups of inhibitors and the amino acid residues present at important subsites of RNase A. The study also established that the free rotating "CH2X" arms of 1,4,5-TTs provided a unique shape to accommodate the molecule within the active site cleft. A fairly good idea about the structure activity relationship (SAR) was obtained by correlating experimentally determined Ki values and the corresponding docking poses. This study reports an unconventional class of non-sugar, non-nucleosidic 1,4,5-TT based competitive inhibitors of RNase A.