1,4,5-Trisubstituted-1,2,3-triazoles (1,4,5-TTs) constitute a special class of functionalized triazoles. This paper reports the design of ribonuclease A (RNase A) inhibitors based on the 1,4,5-TT structural motif. The “catalytic triad” present at the active site of RNase A is responsible for the cleavage of the 5′- phosphodiester bond; amino acid residues His12, Lys41 and His119 constituting this triad provides a positively charged environment at the physiological pH. 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 on the RNase A enzyme by performing qualitative and quantitative biophysical studies. All the “CH2S” and “CH2O” linked acid derivatives (6a-e, 6f′-g′, 6h) & (8a-e, 8f′-g′, 8h) exhibited significant competitive inhibition with inhibition constant values (Ki) ranging from 9 – 34 μM determined by the steady state enzyme kinetics. Especially, uracil based thioglycolic acid (6h) and carboxylic acid based oxy acetic acid (8g′) derivatives were found to be the most promising inhibitors with Ki values 9.9 ± 0.7 and 15.6 ± 0.6 μM, respectively. Moreover, from molecular docking studies, sufficient number of hydrogen bonding interactions were identified with amino acid residues present at important subsites of RNase A and it was revealed that free rotating “CH2X” arms of 1,4,5-TT substituent may provide a unique shape to accommodate the molecule within the active site cleft. Docking results showed a good correlation with experimentally determined Ki values. This study identified an unconventional class of non-sugar, non-nucleosidic competitive inhibitors of RNase A.