Here, we present the development of a pyranopyrazole-based chemosensor (P1) that serves as a reliable colorimetric chemosensor for the identification of biothiols, namely glutathione (GSH), homocysteine (Hcy), and cysteine (Cys). We extensively studied the interaction of P1 with several amino acids (Cys, Ala, His, Ile, Leu, Lys, Ser, Trp, Val, Pro, Phe, Arg) and other biothiols (Hcy and GSH) using colorimetric methods and UV-visible spectroscopy. The results indicated that P1 endures a visible color change in the presence of biothiols, rendering it a practical tool for detection. Time-dependent studies demonstrated that P1 can detect biothiols within seconds, while pH studies verified its efficacy across a wide pH range (4-8), emphasizing its adaptability to a variety of applications. The Benesi-Hildebrand (B-H) method was used to determine the binding constants (Ka) of P1 with Cys, Hcy, and GSH. The respective values obtained were 0.762 × 104 M-1, 0.51 × 104 M-1 and 0.01 × 104 M-1. The determination of the limit of detection (LOD) for Cys, Hcy, and GSH yielded values of 2.62 × 10-7 M, 3.26 × 10-7 M and 1.37 × 10-5 M, correspondingly. Further, the practical usefulness of chemosensor P1 was shown in both solid-state and real sample analysis, encompassing human blood plasma as well as commercially available samples of Cys and GSH. The successful identification of biothiols in these samples underscores the potential of P1 as a reliable chemosensor for clinical diagnostics.