The AlphaLISA® assay is a homogeneous immunoassay alternative to classical ELISA. AlphaLISA assays were originally utilized to detect analytes in cell culture supernatants or serum/plasma samples. More recently, AlphaLISA has been applied to a wider variety of biological matrices including lysates from cultured cells, or fluids and tissue homogenates from animals. We report the development of an assay to measure active caspase-3 in cell lysates from both suspension and adherent cell models (Jurkat and HeLa, respectively). For compound screening, cells are treated with test material and subsequently lysis buffer is added. After a short incubation, target-specific AlphaLISA reagents are directly added, providing a highly efficient all-in-one-well assay format. Under optimal conditions, signal to background (S/B) values up to 17 and Z’ values up to 0.8 were obtained with staurosporine-treated Jurkat cells. Assays were also developed for biological samples derived from rodents or humans. Quantitation of analytes in animal tissue extracts or biological fluids requires an appropriate diluent so that samples can be accurately extrapolated from the calibration curve. Following optimization of the assay, recovery of spiked analytes was generally in the range of 70 to 130%. As examples, mouse interleukin 6 was measured in bronchioalveolar lavage fluid (BALF) at a level of 20 pg/mL, human amyloid beta 1-42 peptide was measured in cerebrospinal fluid (CSF) at a level of 0.3 ng/mL, and mouse vascular endothelial growth factor (VEGF) was detected in lung homogenates at a level of 1 ng/mL. In general, a major advantage of using AlphaLISA for analysis of biological samples is that sample volumes as low as 2.5 µL can be utilized. Also, the absence of wash-steps greatly reduces the assay time and improves the reproducibility of the data.